Isolation of nucleic acids on surfaces

ABSTRACT

New processes and equipment to isolate and purify nucleic acids on surfaces are provided. The invention focuses on processes which use surfaces, for example, porous membranes, on which the nucleic acids are immobilized in a simple manner from the sample containing the nucleic acids and can be released again by way of simple procedural steps, whereby the simple performance of the process according to the invention makes it possible to perform the processes specifically in a fully automatic manner. An additional aspect of the present invention focuses on binding the nucleic acids to an immobile phase, especially to a membrane, in such a way and manner, that they can be released without difficulty during an additional reaction stage from this phase and, if desired, can be used in other applications, such as restriction digestion, RT, PCR or RT-PCR, or in any of the suitable analyses or enzyme reactions mentioned in the disclosure. Special isolation devices are provided that can be used to carry out the processes according to the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of pendingInternational application no. PCT/EP99/02664, filed Apr. 20, 1999 anddesignating the United States, and of pending International applicationno. PCT/EP98/06756, filed Oct. 23, 1998 and designating the UnitedStates and claiming priority to German application DE 19746874.8, filedOct. 23, 1997.

FIELD OF THE INVENTION

[0002] This invention concerns new processes for the isolation andpurification of nucleic acids on surfaces.

BACKGROUND OF THE INVENTION

[0003] It has been known for a long time that the genetic origin andfunctional activity of a cell can be determined and studied byexamination of its nucleic acids. Methods of analyzing nucleic acidspermit direct access to the cause of cell activity. Such methods aretherefore potentially superior to indirect conventional methods such asdetecting metabolic products. For that reason a large expansion in thenumber of nucleic acid analyses can be expected in the future. Forinstance, molecular biological analyses are already used in many areas,for example, in medical and clinical diagnostics, in pharmacology forthe development and evaluation of medications, in analysis of foodstuffsas well as monitoring food manufacturing and food inspection, in theagricultural business for breeding useful plants and animals, inenvironmental analysis, and in many research areas, including, forexample, paternity analyses, tissue typing, identification of geneticdiseases, genome analyses, molecular diagnostics, such as theidentification of infectious diseases, transgenic research, basicresearch in the area of biology and medicine, as well as in numerousrelated areas.

[0004] Through RNA analysis, especially mRNA in cells, gene activity canbe determined directly. The quantitative analysis of transcript patterns(mRNA patterns) in cells, by way of modem molecular biology methods,such as, e.g., real-time reverse transcriptase PCR (“Real-Time RT-PCR”)or gene expression chip analyses, permit for example the recognition ofdefectively expressed genes, through which many types of disorders,e.g., metabolic diseases, infections or the generation of cancer, may berecognized. Analysis of DNA from cells by way of molecular biologicalmethods, such as, e.g., PCR, RFLP, AFLP or sequencing, permits forexample the assessment of genetic defects in or the determination of theHLA type as well as of other genetic markers.

[0005] The analysis of genomic DNA and RNA is also utilized to directlyprove the existence of infectious stimuli, such as viruses, bacteria,etc.

[0006] In this connection, a general difficulty exists in the fact thatbiological and/or clinical samples must be prepared in such a way thatthe nucleic acids contained therein can be utilized directly in theanalytical method in question. It is especially important that thenucleic acids be provided in good yield, that the recovered nucleicacids be of high quality, and that there be high reproducibility, inparticular where there are a greater number of samples, in which casethe analysis should be capable of being conducted automatically.

[0007] The state of the art already includes many processes for thepurification of DNA. For example, it is known how to purify plasmid DNAfor the purpose of cloning and other experimental processes. See, e.g.,the method of Birnboim, Methods in Enzymology, 100:243 (1983). In thisprocess, a cleared lysate of bacterial origin is exposed to a cesiumchloride gradient and centrifuged for a period of 4 to 24 hours. Thisstep is usually followed by the extraction and precipitation of the DNA.This process is associated with the disadvantages that it is veryapparatus-intensive, and it takes a great deal of time, is expensive torun and cannot be automated.

[0008] Other methods in which cleared lysates are used to isolate DNAare based on ion-exchange chromatography (e.g., Colpan et al., J.Chromatog., 296:339 (1984)) and gel filtration (e.g., Moreau et al.,Analyt. Biochem., 166:188 (1987)). These processes are primarilyalternatives to the cesium chloride gradients; however they require anextensive solvent supply system, and a precipitation of the DNAfractions is necessary, since these usually contain salts in highconcentrations and are extremely diluted solutions.

[0009] Marko et al., Analyt. Biochem., 121:382 (1982), and Vogelstein etal., Proc. Nat. Acad. Sci., 76:615 (1979), have found that if the DNAfrom extracts containing nucleic acids is exposed to high concentrationsof sodium iodide or sodium perchlorate, only DNA will adhere to glassscintillation tubes, fiberglass membranes or fiberglass sheets that havebeen finely ground by mechanical means, while RNA and proteins will not.The DNA that has been bound in this manner can be eluted, for example,with water.

[0010] For example, in international publication WO 87/06621, theimmobilization of nucleic acids on a PVDF membrane is described.However, the nucleic acids bound to the PVDF membrane are not eluted inthe next step; instead the membrane, together with all the bound nucleicacids is introduced directly into a PCR reaction. Finally, in thisinternational patent application and in the other literature, it isstated that hydrophobic surfaces or membranes must in general be wettedbeforehand with water or alcohol, in order to be able to immobilize thenucleic acids with yields that are satisfactory.

[0011] On the other hand, for a number of modern applications, such as,for example, the PCR, reversed transcription PCR, SunRise, LCR,branched-DNA, NASBA, or TaqMan technologies and similar real-timequantification methods for PCR, SDA, DNA and RNA chips and arrays forgene expression and mutation analyses, differential display analyses,RFLP, AFLP, cDNA synthesis or substractive hybridization, it isabsolutely necessary to be able to release the nucleic acids directlyfrom the solid phase. In this connection, WO 87/06621 teaches that,while the nucleic acids can indeed be recovered from the membranes usedin the process, this recovery is fraught with problems and is far fromsuited to the quantitative isolation of nucleic acids. In addition, thenucleic acid obtained in this manner is, comparatively, extremelydiluted, which makes subsequent isolation and concentration stepsabsolutely necessary.

SUMMARY OF THE INVENTION

[0012] According to the present invention, all aqueous or othersolutions of nucleic acids, as well as all materials and all samplescontaining nucleic acids, as well as biological samples and materials,foodstuffs, etc. are defined as “nucleic acid samples”. In the sense ofthe present invention, a sample or a material containing a nucleic acidis defined as a nucleic acid sample and/or a sample preparation whichcontains the nucleic acids in question. Biological material and/orbiological samples in this connection include, e.g., cell-free samplematerial, plasma, body fluids—such as for example, blood, sputum, urine,feces, sperm, cells, serum, leucocyte fractions, crusta phlogistica,smears; tissue samples of any type, tissue parts and organs; foodstuffsamples which contain free or bound nucleic acids or nucleicacid-containing cells; environmental samples which contain free or boundnucleic acids or nucleic acid-containing cells, plants and parts ofplants, bacteria, viruses, yeasts and other funghi, other eukaryotes andprokaryotes, etc., as they are published, e.g., in the European patentpublication No. EP 743 950 A1, which is incorporated herein byreference, or free nucleic acids as well. In the sense of the presentinvention, nucleic acids comprise all types of nucleic acids, such as,e.g., ribonucleic acids (RNA) and desoxyribonucleic acids (DNA), in alllengths and configurations, such as double strands, single strand,circular and linear, branched, etc.; monomer nucleotides, oligomers,plasmids, viral and bacterial DNA and RNA, as well as genomic or othernon-genomic DNA and RNA from animal and plant cells or other eukaryotes,tRNA, mRNA in processed and non-processed form, hn-RNA, rRNA and cDNA aswell as all other nucleic acids that can be envisioned.

[0013] For the reasons stated above, the processes known from the stateof the art do not constitute—particularly with regard to automation ofthe process for obtaining nucleic acids—a suitable starting point for anisolation of nucleic acid that is as simple and quantitative as possiblefrom the point of view of process engineering. The purpose of thisinvention is therefore to overcome the disadvantages of the processesknown from the state of the art for the isolation of nucleic acids andto provide a process and method which are capable of being applied orcarried out without substantial technical expenditure.

[0014] According to the present invention, the aforementioneddisadvantages are solved by the processes, isolation and/or reactiondevices uses, automatic apparatus kits according to the description,drawings and claims below.

[0015] In addition, the invention focuses on processes which make use ofsurfaces, e.g., porous membranes, on which the nucleic acids can beeasily immobilized from the sample containing the nucleic acids, and canagain be released by way of similarly easy steps of the process, wherebythe simple performance of the process according to the invention makesit possible to specifically carry out the process in a fully automatedmanner.

[0016] Another purpose of this invention is, in particular, to bindnucleic acids to an immobile solid phase—especially to a membrane—insuch a manner that in a subsequent reaction step they can be releasedimmediately from this phase and, if desired, used in other applications,such as, for example, restriction digest, RT, PCR or RT-PCR, as well asany other suitable analytical or enzymatic reaction named above.

[0017] Within the scope of the present invention, a surface is definedas any microporous separating layer. This may also directly rest on asubstratum and therefore only be accessible from one side or be standingfreely in space. Within the meaning of the present invention a membraneis defined as a separating layer which is accessible from both sideswhen it does not rest with its entire surface area on an impenetrablesubstratum but is entirely free or is only supported at single points.

[0018] Within the meaning of the present invention, isolation is definedas any accumulation of nucleic acids, in which the concentration ofnucleic acids is increased and/or the portion of non-nucleic acids in asample preparation and/or sample is reduced.

[0019] The invention provides a process to isolate nucleic acidsincluding the following steps:

[0020] applying at least one nucleic acid sample to a membrane;

[0021] immobilizing the nucleic acids on the membrane;

[0022] releasing the immobilized nucleic acids from the membrane; and

[0023] removing the released nucleic acids through the membrane,

[0024] whereby the membrane contains nylon, polysulfone,polyethersulfone, polycarbonate, polyacrylate, acrylic copolymer,polyurethane, polyamide, polyvinylchloride, polyfluorocarbonate,polytetrafluoroethylene, polyvinylidene fluoride,polyethylenetetrafluoroethylene-copolymerisate, polybenzimidazole,polyethylene-chlorotrifluoroethylene-copolymerisate, polyimide,polyphenylene sulfide, cellulose, cellulose-mix-ester,cellulose-nitrate, cellulose-acetate, polyacrylnitrile,polyacrylnitrile-copolymers, nitrocellulose, polypropylene and/orpolyester.

[0025] Other membranes also, such as those mentioned below in thepresent description, may be used for processes according to theinvention.

[0026] Preferably the loading process takes place from the top and theremoval process is carried out in a downward direction; however,flow-through processes, for example, can be envisioned in which ahorizontal column is loaded from one side with a solution containingnucleic acid, which, after immobilization of the nucleic acids,penetrates through the membrane and can be removed at the other end ofthe column.

[0027] Preferably, the membrane is situated in a container, e.g., thecolumn mentioned above or any elongated container having an inlet and anoutlet, wherein the membrane stretches across the entire diameter of thecontainer.

[0028] The membrane may be coated so as to render it hydrophobic orhydrophilic.

[0029] Isolation processes to date, especially in isolation columns,function with relatively thick membranes and/or fleeces in order toachieve a complete isolation of the nucleic acids. When the solution issuctioned through the membrane, however, a relatively large, so-calleddead-space-volume, i.e., the volume of the membrane, is generated fromwhich the nucleic acids can only be recovered by way of a largerquantity of an elution buffer. This, however, causes the nucleic acidsto be more diluted after the elution, which is undesirable ordisadvantageous for many applications. For this reason, a preferredembodiment of the invention uses a membrane which is less than 1 mmthick, preferably less than 0.5 mm, and most preferably less than 0.2mm, e.g., 0.1 mm thick.

[0030] The invention furthermore involves a process to isolate nucleicacids with the following steps:

[0031] applying at least one nucleic acid sample to a surface;

[0032] immobilizing the nucleic acids on the surface; and

[0033] releasing the immobilized nucleic acids from the surface with anelution agent.

[0034] This process is characterized in that the release takes place ata temperature whose upper limit is 10° C. or less and whose lower limitis at the freezing point of the elution agent to be used for suchrelease, so that the elution agent does not freeze. Therefore thefollowing inequation applies: 10° C.≧T≧T_(S, EM,) in which T is therelease temperature and T_(S, EM) is the freezing point of the elutionagent. We have discovered that, contrary to widespread opinion, arelease of the nucleic acids near the freezing point of the elutionagent is quite possible. Such an elution at low temperature even has theunexpected advantage that the nucleic acids are treated more gently andthat the activity from any nucleases (DNases or RNases) still present inthe sample drops practically to nothing near the freezing point, so thatdegradation of the nucleic acids is reduced or completely prevented.

[0035] Accordingly, the temperature during elution should preferably beeven lower, e.g., at less than 5° C. The lower limit may also be at 0°C. or −5° C., if the specimen is still liquid at this temperature, basedon its ion content. The upper temperature limit should if possible alsobe low, e.g., at about 5° C.

[0036] The process according to the invention therefore requires coolingof the elution buffer and may require cooling of any additionalsolutions used, as well as cooling of the isolation device if necessary.Since cooling cannot always be guaranteed in a reliable manner,especially during examinations performed in the field, e.g., whenscreening human samples in developing countries, the present inventionalso provides an isolation device which allows isolation of nucleicacids at low temperatures independent from any external cooling. Forsuch situations, the instant invention provides an isolation device toisolate nucleic acids having at least an upper part with a top opening,a bottom opening and a membrane, which is located at the bottom openingand which fills the entire diameter of the upper part; a bottom partwith an absorbent material; and a collar surrounding the upper part, atleast in the area of the membrane, which contains a coolant. The collarcontaining the coolant allows cooling of the membrane and the solutionsplaced on the membrane such as the lysate, washing buffer and elutionbuffer at low temperatures, so that the final elution can take place ina reliable manner within the desired temperature range near the freezingpoint of the elution buffer.

[0037] In an embodiment of this isolation device, the collar has twocompartments, which are separated from one another by a mechanicallydestructible or frangible separation wall, with each of the compartmentscontaining a solution and in which upon mixing of both solutions afterdestruction of the separating wall, the coolant is generated. Theseparating wall can be destroyed by the user, e.g., by pressing againstthe external collar wall, e.g., at points provided for such purpose, andthus causing the separating wall to tear. Suitable solutions to fill thecompartments are familiar to practitioners in the area of chemicalcooling technology. These may be adjusted to the desired temperaturesand to the outside temperatures expected when using the isolationdevice.

[0038] When recovering nucleic acids from biological samples, such asthe samples indicated above, it is often necessary to make a lysate thecells or secretions first, in order to be able to reach the nucleicacid. The lysates thus produced may also contain large amounts ofundesirable substances in addition to the nucleic acids, such asproteins or fats. If the content of such substances in a lysate is toohigh, the membrane may become clogged when the lysate is applied, whichreduces the efficiency of the nucleic acid isolation and which reducesthe permeability of the membrane during washing or elution. In order toavoid this undesirable effect, the invention provides a process in whichundesirable substances are removed before they reach the membrane.

[0039] In preferred embodiments, the process according to the inventionto isolate nucleic acids comprises the following steps:

[0040] adjusting at least one nucleic acid sample to binding conditionswhich allow immobilization of the nucleic acids contained in at leastone of the nucleic acid samples on a surface;

[0041] applying at least one nucleic acid sample to the surface; and

[0042] immobilization of the nucleic acids on the surface,

[0043] characterized in that before and/or after adjusting the bindingconditions, a pretreatment is applied.

[0044] The pretreatment may, for instance, take place by salting out orby filtration, centrifugation, enzymatic treatment, temperature effect,precipitation and/or extraction of the nucleic acid solution and/orbinding contaminants of the nucleic acid solution to surfaces. Thepre-treatment may also involve mechanical disruption or homogenizing thenucleic acid solution, if it is for example the lysate of a biologicalsample.

[0045] The binding conditions that were adjusted may permit theimmobilization of RNA and/or DNA in this case.

[0046] A pre-treatment may be necessary especially in cases when oneintends to isolate biological samples with severe contaminants. Thebiological sample may consist of any conceivable material which is usedeither immediately or can be recovered from another biological sample.For instance, this may be blood, sputum, urine, feces, sperm, cells,serum, leukocyte fractions, crusta phlogistica, smears, tissue samples,plants, bacteria, funghi, viruses and yeasts, as well as all other typesof biological samples mentioned above.

[0047] The process according to the invention may be used to itsgreatest advantage if the biological sample contains a large amount ofundesirable substances.

[0048] After immobilization of the nucleic acids from the pre-treatednucleic acid sample, the usual isolation steps can be followed, i.e.:

[0049] releasing the immobilized nucleic acids from the surface;

[0050] recovering the nucleic acids released from the surface.

[0051] A special advantage of the isolation process according to theinvention concerns the fact that it may be connected with chemicalreactions, to which the nucleic acids are subjected directly on thesurface. A variety of analytical techniques for nucleic acids maytherefore be used with the nucleic acids isolated on the surface. Inthis case it is possible to again release the nucleic acids from thesurface prior to the reaction in order to guarantee their freeaccessibility. Alternatively, a suitable reaction may also be performedwith the nucleic acids which are directly bound on the surface.

[0052] Accordingly, one aspect of the invention involves a process witha pre-treatment, as outlined above, which is characterized in that thefollowing step preferably takes place at least once after the releasestage:

[0053] performing at least one chemical reaction with the nucleic acids.

[0054] A special advantage of this process lies in the fact that priorto the chemical reaction, no loss resulting from transfer of the nucleicacids from the isolation device to a reaction device occurs, because theisolation and chemical reaction can take place in the same device.

[0055] In an additional aspect not related to pre-treatment, theinvention involves a process to carry out a nucleic acid amplificationreaction with the following steps:

[0056] applying at least one nucleic acid sample to a surface;

[0057] immobilizing the nucleic acids on the surface; and

[0058] performing an amplification reaction with the nucleic acids.

[0059] Especially with the small quantities of material commonly used inamplification reactions or available for use in amplification reactions,it is generally advantageous if the whole reaction sample of nucleicacids can be used in the reaction without any loss from transfer. Thisis especially advantageous for an automated process since all steps canbe carried out in one device. Furthermore, the amount of waste isreduced and the process is faster and more cost-effective.

[0060] The amplification reaction may be an isothermal or anon-isothermal reaction.

[0061] The amplification reaction may, e.g., consist of an SDA-reaction(“strand displacement amplification”), a PCR, RT-PCR, LCR or a TMA or arolling circle amplification.

[0062] A NASBA-reaction is also possible with this process according tothe invention.

[0063] Prior to carrying out the amplification reaction, the nucleicacids may be released from the surface with a reaction buffer, wherebythe eluate is located on or in the membrane. Alternatively, theamplification reaction may be carried out in a reaction buffer that doesnot produce a release of the nucleic acids from the surface.

[0064] This process preferably produces these additional steps:

[0065] if necessary, release of the reaction products from the surface(to the extent these were still bound during the reaction); and

[0066] removal of the released reaction products from the surface.

[0067] Another aspect involves a process to perform chemical reactionswith nucleic acids by way of the following steps:

[0068] applying at least one nucleic acid sample to a surface;

[0069] immobilizing nucleic acids on the surface;

[0070] releasing the immobilized nucleic acids from the surface;

[0071] performing at least one chemical reaction with the nucleic acids;and

[0072] removal of the nucleic acids from the surface without priorimmobilization.

[0073] In this process the nucleic acids are no longer bound(immobilized) to the membrane after the chemical reaction, but removedwithout binding. Although the elimination of such an additional step maycompromise the purity of the removed specimen, it may be preferredbecause it saves time in critical applications and it also simplifiescertain application methods. A wide range of chemical reactions isavailable as a result of the process according to the invention. Withinthe meaning of the invention “chemical reaction” should be defined inthis connection as any interaction of the nucleic acids with othersubstances (with the exception of the surface, since this “reaction”occurs in all processes described herein), i.e., enzymaticmodifications, hybridization with probes, chemical sequencing reactions,pH-value-changes, e.g., for basic depurination of RNA and aciddepurination of DNA, as well as antibody binding and protein binding.Generally, each reaction, whether it concerns the changing of covalentbonds or hydrogen bonds, is included.

[0074] One advantage of the process according to the invention is thepermanent, spatial combination of a volume chamber, in which a greatvariety of processes can take place, and a membrane to which nucleicacids can be bound. Simply put, this combination allows the manipulationof nucleic acids followed by binding to a membrane. This is especiallyadvantageous for automated processes. After binding to the membrane, thenucleic acids are available for additional treatment steps, e.g., asmentioned above, for isolation of highly pure nucleic acids or forperforming chemical reactions with the nucleic acids. An additionalaspect of the invention makes it also possible to immediately subjectthe nucleic acids still bound to the membrane to further analysis, inorder to determine certain properties of the nucleic acids.

[0075] For that reason the invention also involves a process to analyzenucleic acids in an isolation device with the following steps:

[0076] making available an isolation device with a membrane locatedtherein;

[0077] applying at least one nucleic acid sample to the isolationdevice;

[0078] immobilizing the nucleic acids on the membrane;

[0079] leading the fluid components of the sample through the membrane;and

[0080] analyzing at least one property of the nucleic acids on themembrane located in the isolation device.

[0081] After passing the fluid components through the membrane, at leastone chemical reaction as mentioned above can be performed with thenucleic acids in an additional embodiment. This may serve, e.g., toallow the subsequent analysis of the nucleic acids. Examples ofreactions in this context are the hybridization of probes, theradioactive labeling of nucleic acids bound to the membrane or thebinding of specific antibodies. Auxiliary reactions such as stainingnucleic acids, e.g., with intercalating substances such as ethidiumbromide should also be considered as a chemical reaction.

[0082] Various properties of nucleic acids are open to an analysis whilethey are bound to the membrane. They have already been described forconventional membranes without a combined reaction device. Some of theproperties that can be analyzed are the radioactivity of nucleic acidsor their binding affinity for molecules, in which the molecules forexample may be antibodies or dye molecules that bind nucleic acids orare bound to nucleic acids or proteins.

[0083] This process represents a considerable simplification of theanalysis of nucleic acids, since the manipulation of the free membraneis no longer necessary. This is now located in the isolation device.

[0084] An irreversible bond of the nucleic acids to the membrane, e.g.,for subsequent analytical steps is also within the scope of the presentinvention. This long-lasting or irreversible bond permits themanipulation of the membrane and the nucleic acids bound thereon to anextent that is not possible for reversibly bound nucleic acids.

[0085] An additional aspect of the invention focuses on the quantitativeprecipitation of nucleic acids.

[0086] In previously known methods based on anion-exchangechromatography for purification of 100 μg and more plasmid-DNA(hereinafter indicated as “large scale” DNA purification), theplasmid-DNA is eluted in a high saline buffer from the column during thelast step. In order to separate the plasmid-DNA from the salt on the onehand, and to concentrate it on the other, it is precipitated with theaid of alcohol (e.g., isopropanol) and centrifuged in a suitable device.The centrifugation pellet thus obtained is washed with 70% ethanol, inorder to remove the residual traces of salt and is then again subjectedto centrifugation. The pellet from the second centrifugation istypically dissolved in a small amount of low saline buffer and theplasmid-DNA is processed further in this form.

[0087] In addition, the state of the art has proposed processes in whichDNA is added in such a form by adding chaotropic salts to the highsaline buffer so as to cause binding to silica membranes. After acorresponding washing step, the DNA can again be released from themembrane by way of a low saline buffer.

[0088] A similar application is described in a publication (Ruppert etal., Analytical Biochemistry, 230: 130-134 (1995)) in which on a smallscale (isolation of less than 100 μg of plasmid-DNA) DNA precipitatedwith isopropanol is bound to PVDF-membranes with pore sizes of less than0.2 μm, subsequently washed with ethanol and then eluted with TE(Tris-EDTA). However, there is no description of such a method for thelarge scale process.

[0089] The DNA precipitation described with subsequent centrifugation isextremely time-consuming (approx. 1 hour), and furthermore requires theuse of centrifuges. In addition to the time factor for this procedure,the last step described for plasmid preparation is particularly prone toerrors. A partial or complete loss of the DNA-pellet also occursoccasionally. A decisive roll appears to be the type (material) of thecentrifugation device used.

[0090] The use of chaotropic salts (also described) and the subsequentbinding of nucleic acids to silica membranes is also time-consuming;moreover, because of the introduction of chaotropic salts to thepreparation there is the risk of contamination of the finally isolatedDNA.

[0091] The filtration of alcoholic precipitates on a small scale asdescribed above has the disadvantage that the operation cannot betransferred linearly to a large scale process. Conventional membranesonly permit the isolation of small amounts of nucleic acids, as themembranes are quickly saturated with nucleic acids and no longer absorbanything. When the precipitate buffer is removed and washed, a largeportion of the nucleic acids is frequently lost again. In order to avoidthis loss, the invention also involves a process to precipitate nucleicacids by way of the following steps:

[0092] making available an isolation device with at least one membranesituated therein;

[0093] applying a nucleic acid sample to the isolation device;

[0094] precipitation of the nucleic acids contained in the sample withalcohol, so that the nucleic acids are at least bound to a membrane. Theprocess is characterized in that the pore size of at least one membraneis the same or greater than 0.2 micrometers.

[0095] Alcohols considered to perform the process according to theinvention are first of all hydroxyl derivates of aliphatic or acyclicalsaturated or unsaturated hydrocarbons.

[0096] Among the aforementioned hydroxyl compounds, the C₁-C₅ alkanols,such as methanol, ethanol, n-propanol, n-butanol, tert-butanol,n-pentanol or mixtures thereof are preferred. Especially preferred isthe use of isopropanol to carry out the process according to theinvention.

[0097] In this process, the alcohol can be mixed with this solutionbefore or after loading the isolation device with the solutioncontaining the nucleic acid. The volume ratio of the nucleicacid-containing solution to alcohol, especially isopropanol, preferablyis 2:1 to 1:1, most preferably 1.67:1 to 1:1, and for example 1.43:1.

[0098] The surface of the membrane is preferably chosen so that all thenucleic acids contained in the solution can be bound to the membrane.

[0099] The invention also involves the use of membranes with a pore sizeof equal or larger than 0.2 μm to bind the alcohol-precipitated nucleicacids, which may consist of DNA and/or RNA. Especially advantageous isthe use of a 0.45 μm cellulose acetate or cellulose nitrate filterand/or the use of various layers of a 0.65 μm cellulose acetate orcellulose nitrate filter. The procedure can both be used as vacuumfiltration and as pressure filtration.

[0100] The process according to the invention permits a time-savingtransfer of nucleic acids from a high-salt buffer system to a low-saltbuffer system, which is possible without use of complex apparatus. It issuitable as a substitute for the classical alcoholic precipitation ofDNA from a high-salt buffer, which is typically by centrifugation steps.Because of the great effectiveness of the method (minor loss of yield)it is especially suitable as a preparation for a large scale process.Furthermore the process according to the invention does not introduceany additional substances in the already purified nucleic acids. Inaddition, compared to the classical method, susceptibility to errors isless (loss of the centrifugation sediment during the washing cycle isnot possible using the process of the invention).

[0101] Preferably, applying the solution should take place from the topin the various processes explained above. In principle, a wide range ofmethods are available which pass various solutions such as nucleicacid-containing immobilization buffers, washing buffers, eluate, etc.through the membranes.

[0102] This may be achieved through gravity, centrifugation, vacuum,positive pressure (on the loading side), and capillary forces.

[0103] Between the immobilization and the separation step, theimmobilized nucleic acids may be washed with at least one washingbuffer. The washing preferably consists of the following steps for eachwashing buffer:

[0104] applying a predetermined quantity of washing buffer to thesurface, and

[0105] passing the washing buffer through the surface.

[0106] The application and immobilization of the nucleic acids may againconsist of the following steps:

[0107] mixing of the nucleic acid sample with an immobilization buffer;

[0108] applying the nucleic acid sample with the immobilization bufferon the surface, and

[0109] passing the liquid components through the surface in essentiallythe direction of the loading step.

[0110] The processes have the major advantage that they can easily beautomated, so that at least one of the steps can be fully automated inan automatic device. It is also possible to have all steps of theprocesses performed in a pre-arranged sequence by an automaticapparatus. Especially in these cases, but also for manual handling, itis possible that a majority of nucleic acids are simultaneously subjectto isolation. For example, multi-isolation devices may be used in theform of commonly available “multi-well” devices with 8, 12, 24, 48, 96or more single isolation wells.

[0111] The removal of the nucleic acids may take place in two roughlydifferent directions. On the one hand it is possible to feed (pass) the(eluted) nucleic acids that were removed through the membrane and toremove them toward the membrane's side, that is located opposite theside on which the nucleic acid-containing solution and/or the lysate wasplaced. In this case the nucleic acid is removed in the direction of itspassing through the membrane. The other possibility consists of removingthe nucleic acids from the membrane and/or from the surface on the sidewhere they were introduced. The removal then takes place in thedirection opposite to their introduction or “in the same direction”, inwhich they were introduced; in other words, on the side where they wereintroduced. In this case the nucleic acids do not pass through themembrane. In some of the processes according to the invention, removalof the nucleic acids takes place through the membrane in the directionthey were introduced. In the event a process is carried out with asurface that does not have a non-permeable substratum, e.g., a syntheticlayer, the removal can of course only take place in the direction ofintroduction (hence in the opposite direction). For a few processes,however, the substance can be removed in both directions.

[0112] If the nucleic acids are eluted (released) from the surfaceessentially in the opposite direction from the direction in which theywere introduced and immobilized, “the same direction” is essentiallyconsidered each direction with an angle equal or smaller than 180°,compared to the direction of introduction, so that upon elution, thenucleic acids under no circumstances permeate the surface, e.g., amembrane, but are removed from the surface in the direction oppositefrom the loading direction in which they were introduced to the surface.In preferred embodiments, on the other hand, the other buffers, i.e.,those buffers which contain nucleic acids during the loading process,and if required a washing buffer, are suctioned through the surface orotherwise transferred. If the isolation takes place on a membranelocated in a device, whereby the membrane fills the entire diameter ofthe device, the preferred loading method is from the top. In this casethe removal step again occurs upward. FIG. 2 shows an example of afunnel-shaped isolation device, which is loaded from the top and inwhich the removal of the nucleic acids takes place in an upwarddirection.

[0113] It is understood that, in the case of removal in a directionopposite to introduction, other configurations are also imaginable,e.g., removal of the nucleic acids from below. It is possible, forexample, to suction a buffer containing nucleic acids, such as a lysatebuffer from a reaction device directly into an isolation device by wayof a suction installation, so that the nucleic acids will be bound tothe bottom of a membrane in the isolation device. In such a case, theremoval of the nucleic acids from the surface can be carried out, insuch a way that an elution buffer is suctioned up from below and isdrained again downward into a device after separation of the nucleicacids. The removal of the nucleic acids therefore also takes place in adownward direction.

[0114] A lateral removal of the nucleic acids is also possible, e.g., ifa horizontal column with a membrane located therein is loaded with alysate during the flow-through process and the horizontal column issubsequently washed with elution buffers on the side of the membrane towhich the nucleic acids are bound.

[0115] An example for the maximum possible angle of 180° is a slope witha surface suitable to bind nucleic acids along which surface the varioussolutions and/or buffers flow. Like all buffers, the elution buffer alsoarrives from one side and is drained on the other side. In this case,the inflow direction of the buffer and the draining direction of thebuffer with the nucleic acids included therein make an angle of 180°;the removal, however, continues to take place on the same side of thesurface as the immobilization.

[0116] Following the process according to the invention, the samplecontaining nucleic acids described above is added to a solution whichcontains the appropriate salts and/or alcohol(s); subsequently thesample is lysed, if necessary, and the mixture obtained in this manneris led by way of a vacuum, centrifugation, positive pressure, capillaryforces or by way of other appropriate processes, through a poroussurface, whereby the nucleic acids are immobilized on the surface.

[0117] Suitable salts for the immobilization of nucleic acids onmembranes or other surfaces and/or for the lysis of nucleic acid samplesare salts of metal cations, such as alkaline or alkaline earth metals,with mineral acids; especially alkaline or alkaline-earth halides and/orsulfates or phosphates, including the halides of sodium, lithium orpotassium or magnesium sulfate, which are most preferable. Other metalcations, e.g., Mn, Cu, Cs or Al, or the ammonium cation can be used,preferably as salts of mineral acids.

[0118] Furthermore to carry out the process according to the invention,salts having one or more basic functions or even polyfunctional organicacids with alkaline or alkaline-earth metals are suitable. Theseespecially include sodium, potassium or magnesium salts with organicdicarboxylic acids, such as e.g., oxalic, malonic or succinic acids, orwith hydroxy and/or polyhydroxycarboxylic acids, such as, e.g., withcitric acids, preferably.

[0119] The substances indicated above to immobilize the nucleic acids onsurfaces and/or for the lysis of nucleic acid samples may be usedseparately or in mixtures, if this should prove to be more suitable forcertain applications.

[0120] In this connection the use of so-called chaotropic agents hasproved to be particularly effective. Chaotropic substances are able todisrupt the three-dimensional structure of hydrogen bonds. This alsoweakens the intramolecular binding forces which are involved in theformation of spatial structures, such as, e.g., primary, secondary,tertiary or quaternary structures, in biological molecules. Suitablechaotropic agents are well known to those skilled in the art (see,Römpp, Lexikon der Biotechnologie, Publisher H. Dellweg, R. D. Schmidand W. E. Fromm, Thieme Verlag, Stuttgart 1992).

[0121] According to this invention preferred chaotropic substances aresalts from the group of trichloroacetates, thiocyanates, perchlorates,iodides or guanidinium hydrochloride and urea. The chaotropic substancesare then used in a 0.01 to 10 molar aqueous solution, preferably in a0.1 to 7 molar aqueous solution, and most preferably in a 0.2 to 5 molaraqueous solution. In this connection the aforementioned chaotropicagents can be used individually or in combination. Most preferably 0.01to 10 molar aqueous solutions, or 0.1 to 7 molar aqueous solutions, or0.2 to 5 molar aqueous solutions of sodium perchlorate, guanidiniumhydrochloride, guanidinium isothiocyanate, sodium iodide and/orpotassium iodide are used.

[0122] The salt solutions used in the processes according to theinvention for lysis, binding, washing and/or for elution are preferablybuffered. All suitable buffer systems can be considered as buffersubstances, such as, e.g., carboxylic acid buffers, especially citratebuffers, acetate buffers, succinate buffers, malonate buffers as well asglycine buffers, morpholino-propane-sulfone-acids (MOPS) orTris(hydroxymethyl)aminomethane(Tris) in concentrations of 0.001 to 3mol/liter, preferably 0.005 to 1 mol/liter, and most preferably 0.01 to0.5 mol/liter, and particularly preferred 0.01 to 0.2 mol/liter.

[0123] To carry out the process according to the invention, first allhydroxyl derivates of aliphatic or acyclical saturated or unsaturatedhydrocarbons are eligible as alcohols. It is irrelevant whether thesecompounds contain one, two, three or more hydroxyl groups—such aspolyvalent C₁-C₅ alkanols, e.g., ethylene glycol, propylene glycol orglycerin.

[0124] In addition, the alcohols that can be used according to theinvention also include sugar derivates, the so-called aldites, as wellas phenols, e.g., polyphenols.

[0125] Among the aforementioned hydroxy compounds, C₁-C₅-alkanols, suchas methanol, ethanol, n-propanol, tert.-butanol and pentanols, ormixtures of such alcohols, are most preferred.

[0126] Within the meaning of this invention, such substances and/ormembranes which by their chemical nature easily mix with water or absorbwater are considered hydrophilic.

[0127] Within the meaning of this invention, such substances and/ormembranes which by their chemical nature do not penetrate water orvice-versa and which cannot stay dissolved in water are consideredhydrophobic.

[0128] Within the meaning of this invention, any microporous separatinglayer is understood to be a surface. In the case of a membrane thesurface consists of a film made of polymer material. The polymerpreferably consists of monomers with polar groups.

[0129] In a further embodiment of the process according to theinvention, the concept of surface furthermore also comprises a layer ofparticles and/or a granulate as well as fibers such as silica gelfleece.

[0130] When hydrophobic membranes are used in the practice of thisinvention, membranes are preferred which consist of a hydrophilic basicmaterial and which are made hydrophobic by a corresponding chemicalpost-treatment which is known from the state of the art. Membranes suchas commercially available hydrophobic nylon membranes are preferablyused.

[0131] According to the invention membranes that are hydrophobic aregenerally defined as those membranes which are originally hydrophilicmembranes that have been coated with hydrophobical coating agentsmentioned below. Such hydrophobical coating agents coat the hydrophilicsubstances with a thin film of hydrophobic groups, which, e.g., includelonger alkyl chains or siloxane groups. Many suitable hydrophobiccoating agents are known and include, e.g., paraffins, waxes, metallicsoaps, etc., if necessary with additions of aluminum, zirconium salts,quaternary organic compounds, ureic derivates, lipid-modified melamineresins, silicones, zinc organic compounds, glutaric dialdehydes, andsimilar compounds.

[0132] According to the invention suitable hydrophobic membranes alsoare those membranes which are by themselves hydrophobic or which havebeen made hydrophobic and whose basic material may contain polar groups.According to these criteria, e.g., especially hydrophobic materials fromthe following group are suitable for use according to the invention:Nylon, polysulfones, polyether sulfones, cellulose nitrate,polypropylene, polycarbonates, polyacrylates as well as acryliccopolymers, polyurethanes, polyamides, polyvinyl-chloride,polyfluorocarbonates, polytetrafluoroethylene, polyvinylidene fluoride,polyethylene-tetra-fluoroethylene copolymerisates,polyethylene-chlorotrifluoro-ethylene-copolymerisates, or polyphenylenesulfide, as well as cellulose and cellulose-mix esters, celluloseacetate or nitrocellulose as well as polybenzimidazoles, polyimides,polyacryl nitriles, polyacrylnitril-copolymers, hydrophobisized glassfiber membranes, including hydrophobisized nylon membranes which aremost preferable.

[0133] Preferred hydrophilic surfaces include hydrophilic materials perse and also hydrophobic materials which have been hydrophilisized. Forinstance the following substances can be used: hydrophilic nylon,hydrophilic polyether-sulfones, hydrophilic polycarbonates, hydrophilicpolyesters, hydrophilic poly-tetra-fluoroethylenes on polypropylenetissues, hydrophilic polytetrafluoroethylenes on polypropylene fleece,hydrophilisized polyvinylidene fluoride, hydrophilisizedpolytetrafluoroethylenes, hydrophilic polyamides, nitrocellulose,hydrophilic polybenzimidazoles, hydrophilic polyimides, hydrophilicpolyacryl-nitriles, hydrophilic polyacrylnitril-copolymers, hydrophilicpolypropylene, cellulose nitrate, cellulose-mix-ester and celluloseacetate.

[0134] The membranes described above are already known in the art,partially for their use in nucleic acid binding, but not yet in thecontext of the invention. A series of materials for this particular useis, however, not known from the state of the art. The extensive trialsdisclosed herein have demonstrated that there are additional membranesthat are suitable to bind nucleic acids.

[0135] The present invention therefore also involves the use ofcellulose acetate, non-carboxylized, hydrophobic polyvinylidenefluoride, or massive hydrophobic poly-tetra-fluorethylene as a materialon which to precipitate and isolate nucleic acids. In this context, theterm “massive” denotes a material which generally consists of thecorresponding compound and is neither coated nor applied as a coating ona carrier material.

[0136] The material may be used as a membrane, as granulate, as fibersor in other suitable forms. The fibers may, e.g., be configured asfleece and the granulate may be pressed as a grid.

[0137] The membranes used in the process described above according tothe invention (with the exception of isopropanol precipitate) forinstance have a pore diameter of 0.001 to 50 μm, preferably 0.01 to 20μm, and most preferably a pore size of 0.05 to 10 μm. In case thenucleic acids are precipitated with isopropanol according to the processdescribed above, the pore size must be greater than 0.2 μm.

[0138] The salts or alcohols described above or the phenols orpolyphenols may also be considered as washing buffers. Detergents andnatural substances in the broadest sense of the word, such as albumin,or milk powder may also be used for the washing steps. The addition ofchaotropic substances is also possible. Polymers as well as detergentswith dissolving abilities and similar materials may also be added. Thewashing buffers and the substances contained therein should at any rategenerally be able to bind undesirable contaminants, to dissolve them orto react with them, so that these contaminants or their decompositionproducts can be removed jointly with the washing buffer.

[0139] The temperatures during the washing stage typically range fromabout 10° to 30° C., preferably at room temperature, although higher orlower temperatures may also be applied successfully. When elution isperformed at a low temperature, e.g. 2° C., one should not forget toalso cool the washing buffer in order to pre-cool the temperature of theisolation device and the surface and/or membrane to the desiredtemperature. One application for low temperatures is cytoplasmaticlysis, during which the cell nuclei remain undamaged. Highertemperatures of the washing buffers on the other hand cause betterdissolution of the contaminants to be washed out.

[0140] Suitable eluting agents for the purposes of the invention arewater or aqueous salt solutions. Buffer solutions that are known fromthe state of the art are used as salt solutions, such asmorpholinopropane sulfonic acid (MOPS),tris(hydroxymethyl)aminomethane(TRIS),2-[4-(2-hydroxyethyl)piperazino]ethane sulfonic acid (HEPES) in aconcentration from 0.001 to 0.5 moles/liter, preferably 0.01 to 0.2moles/liter, most preferably 0.01 to 0.05 molar solutions. Alsopreferred for use are aqueous solutions of alkaline or alkaline-earthmetal salts, in particular, their halogenides, for example, including0.001 to 0.5 molar (preferably 0.01 to 0.2 molar, most preferably 0.01to 0.05 molar) aqueous solutions of sodium chloride, lithium chloride,potassium chloride or magnesium chloride. Also preferred for use aresolutions of salts of the alkaline or alkaline-earth metals withcarboxylic or dicarboxylic acids, e.g., oxalic acid or acetic acid, orsolutions of sodium acetate or sodium oxalate in water, e.g., in theconcentration range mentioned above, such as 0.001 to 0.5 molar,preferably 0.01 to 0.2 molar, most preferably from 0.01 to 0.05 molar.

[0141] The addition of subsidiary compounds such as detergents or DMSOis also possible. If a chemical reaction must be carried out with theeluted nucleic acids, either directly on the membrane or in anotherreaction device, it is also possible to add such substances or othersubsidiary compounds which are to be used in the reaction to the elutionbuffer. For instance, the addition of DMSO in low concentrations iscustomary in many reactions.

[0142] After a chemical reaction with the nucleic acids, these can alsobe eluted with the reaction buffer. For instance, the nucleic acids canbe eluted with the reaction buffer or the reaction master mix after aSDA- or a NASBA-reaction.

[0143] Most specifically, pure water is the preferred elution agent,e.g., demineralized, bi-distilled, or ultra pure millipore water.

[0144] The elution can, for example, be carried out successfully attemperatures from below 0° C. to 90° C., e.g., from 10° to 30° C. or athigher temperatures. It is also possible to elute with water vapor. Thelower limit of the elution temperature is, as explained above, thefreezing point of the elution buffer.

[0145] Based on the smooth executability of the processes according tothe invention which can also be performed “in the field”, i.e., outsideof established laboratory installations and therefore without extensiveelectrically powered equipment, the invention also involves thepreparation of isolation devices with which the process according to theinvention can be carried out with a minimum of additional subsidiarymaterials. For this, a reaction device can be used which contains amembrane. This can be brought into contact with an absorbent material,such as a sponge, in order to absorb the various buffers used throughthe membrane. The sponge acts therefore as a combination vacuum pump orcentrifuge in conjunction with a waste collector. In order to recoverthe eluate, contact of the absorbent material with the membrane iseliminated, so that the eluate cannot be lost, but instead can beremoved or studied further.

[0146] In this aspect, the invention specifically involves an isolationdevice to isolate nucleic acids with at least a cylindrical upper partwith a top opening, a bottom opening and a membrane which is located atthe bottom opening and fills the entire diameter of the upper part; isequipped with a bottom part containing an absorbent material; and amechanism for the connection between the upper and lower parts, inwhich, after the connection has been made, the membrane is in contactwith the absorbent material, and when the connection is not made, themembrane is not in contact with the absorbent material.

[0147] Preferably, the bottom or lower part is a cylinder with the samediameter as the upper part. In this manner, a simple tube is obtainedhaving essentially a constant diameter, which can be handled in the sameway as traditional reaction devices. Especially if the upper part or theupper part plus lower part create a tube which can be placed in reactiondevice holders, such as those used in laboratories, this effect can beachieved. The mechanism can be a connection which allows a spatialseparation of the upper and lower parts, for example a bayonet socket, aplug-in socket or a threaded end. A bayonet socket has the advantagethat it is easier to lock and unlock, whereas the threaded connectionallows for a better, more watertight connection of the upper and lowerparts. Alternatively, a predetermined breaking point can be providedbetween the upper and lower parts, which at least allows for theone-time separation of both parts and which can be manufactured at avery low price. Alternatively, the connection can also be a slidingmechanism which can be slid between the absorbent material and themembrane. In this embodiment, a separation of membrane and absorbentmaterial can be achieved as well.

[0148] To increase the processing capacity and to be able to carry outthe process according to the invention even more economically, it isalso possible to modify the isolation device according to the inventiondescribed above in such a way that various upper parts are placed on abottom part. The bottom part can serve simultaneously as a holder of theassembly and in addition have such dimensions that a variety ofisolation processes, at least more than mere connections for the upperand lower parts, are available, and can be carried out before thesuction capacity of the absorbent material in the bottom part isexhausted.

[0149] The absorbent material in the lower part may contain a spongeand/or a granulate. The granulate can consist of a superabsorbentmaterial, as is known by those skilled in the art of absorptiontechnology (e.g., for hygiene-related items).

[0150] The invention similarly involves utilization of this isolationdevice according to the invention for the analysis of properties ofnucleic acids and to isolate nucleic acids.

[0151] With respect to the separate stages, the processes according tothe invention are typically carried out as follows:

[0152] When starting from biological samples, they must first besubjected to lysis in the appropriate buffers. Additional processes toachieve lysis may be needed, e.g., a mechanical action, such ashomogenization or ultrasound, enzymatic reaction, temperature changes oradditives. In case it is required or desirable, a pre-treatment canfollow this lysis in order to remove debris from the lysate.Subsequently, in case this has not happened yet, the conditions in thelysate are adjusted, so that immobilization of the nucleic acids on thesurface can take place. Even after adjustment of the binding conditions,a pre-treatment step can follow cumulatively or alternatively to theabove pre-treatment step.

[0153] This pretreated lysate of the sample used for the recovery ofnucleic acids or the originally free nucleic acid(s)—if one did notstart from a biological sample—is/are pipetted, for example, in a(plastic) column, in which the hydrophobic membrane is fastened, forexample, on the floor. It is more efficient if the membrane is fastenedto a grid, which serves as a mechanical support. The lysate is thenconducted through the membrane, which can be achieved by applying avacuum at the outlet of the column. The transport can also beaccomplished by applying positive pressure to the lysate. In addition,as mentioned above, the transport of the lysate can take place bycentrifugation or by the effect of capillary forces. The latter can beproduced, for example, with a sponge-like material which is introducedbelow the membrane and is in contact with the lysate or filtrate. In thecase of centrifugation, the isolation device open at the bottom may beused in a collection tube for the flowthrough liquid.

[0154] The washing stage included in the preferred embodiments can takeplace if the washing buffer is transported through the surface of themembrane or is remaining on the same side of the surface as the nucleicacids. If the washing buffer is transported or suctioned through, thiscan take place in different ways, e.g., by a sponge located on the otherside of the membrane, a suction or positive pressure mechanism or bycentrifugation or gravity.

[0155] The advantage of a configuration utilizing an absorbent, possiblyspongy material is that it provides a simple, secure and handy means fordisposing of the filtrate, in this case only the sponge, which by thattime is more or less saturated with the filtrate and needs to bereplaced. At this point it is clear that the column can be operatedcontinuously or also in a batch-like manner, and that both modes ofoperation can be filly automated, until the membrane is saturated withnucleic acids. In the last stage, if required, the elution of thenucleic acids takes place, which for example can be pipetted or liftedfrom the membrane or can be removed upward in another way, if no in situanalysis of the nucleic acids that are still bound is to be performed.

[0156] The desired nucleic acids are present in very small volumes ofbuffers with no or low salt concentrations, which is a great advantagefor all molecular biological analyses, since it is always desirable tohave pure nucleic acids in high concentrations and in the smallestvolumes possible. In order to obtain the smallest possible volumes ofeluate, it is especially preferred to use as surfaces those membranesthat are as thin as possible, so that only very little liquid canaccumulate in them.

[0157] Furthermore, the present invention offers the advantage that inthe case of a vertical configuration of the device (where the membraneis placed in a horizontal direction) the volume located above themembrane can be used as a reaction chamber. Hence, it is possible, forexample, after isolation and removal of the nucleic acids recoveredaccording to the process of the invention, to not remove themimmediately but to leave them in the isolation device and to subjectthem to a molecular biological application, such as restriction digest,RT, PCR, RT-PCR, in vitro transcription, NASBA, rolling circle, LCR(ligase chain reaction), SDA (strand displacement amplification) orenzyme reactions, such as RNase- and DNase-digestion for the completeremoval of any of the nucleic acids that are not wanted, to bind thenucleic acids resulting from these reactions again to the membraneaccording to the process according to the invention or to leave them inthe supernatant, if necessary to wash them as described, andsubsequently to elute them, to isolate and/or analyze them, e.g., by wayof chromatography, spectroscopy, fluorometry, electrophoresis, orsimilar measurements.

[0158] The nucleic acids isolated according to the invention are free ofenzymes that degrade the nucleic acids and have such a high purity thatthey can immediately be used and processed in the greatest variety ofways.

[0159] The nucleic acids produced according to the invention can be usedfor cloning and as substrates for a great variety of enzymes, such as,e.g., DNA-polymerases, RNA-polymerases such as, e.g., T7-polymerase orT3-polymerase, DNA-restriction enzymes, DNA-ligase, reversetranscriptase and others.

[0160] The nucleic acids produced by the processes of the invention areespecially suitable for amplification, especially for PCR, stranddisplacement amplification, rolling circle processes, ligase chainreaction (LCR), SunRise, NASBA and similar processes.

[0161] The processes according to the invention are furthermoreextremely suitable to produce nucleic acids for their use indiagnostics, e.g., in food analysis, in toxicological examinations, inmedical and clinical diagnostics, in diagnostics of germs, geneexpression analysis, and in environmental analysis. The processes areespecially suitable for a diagnostic process, which is characterized inthat the nucleic acids purified by way of the processes according to theinvention are amplified in a subsequent step, and the nucleic acids thatare thus amplified are detected subsequently and/or simultaneously (see,e.g., Holland et al., 1991, Proc. Natl. Acad. Sci., 88: 7276-7280; Livaket al., 1995, PCR Methods Applic., 4: 357-362; Kievits et al., 1991, J.Virol. Meth., 35: 273-286; Uyttendaele et al., 1994, J. Appl.Bacteriol., 77: 694-701).

[0162] Moreover, the processes according to the invention are especiallysuitable to produce nucleic acids which, in a subsequent step, aresubjected to a signal amplification step based on a hybridizationreaction, which is specifically characterized by the fact that thenucleic acids produced in the process according to the invention arebrought into contact with “branched nucleic acids”, especially branchedDNA and/or branched RNA and/or corresponding dendritic nucleic acids andthe signal that is generated is detected, as described in the followingliterature (e.g., Bresters et al., 1994, J. Med. Virol., 43(3): 262-286;Collins et al., 1997, Nucl. Acids Res., 25(15): 2979-2984).

[0163] An example of automation of a process according to the inventionis explained below and examples to perform the process with differentsurfaces and nucleic acids are also described. In this descriptionreference is made to the attached figures which illustrate thefollowing.

BRIEF DESCRIPTION OF THE DRAWINGS

[0164]FIG. 1 shows automatic equipment suitable to perform the processaccording to the invention in a stylized graph.

[0165]FIG. 2 shows a first embodiment of an isolation device andcollector to perform the process according to the invention.

[0166]FIG. 3 shows a second embodiment of an isolation device andcollector to perform the process according to the invention.

[0167]FIG. 4 shows a third embodiment of an isolation device andcollector to perform the process according to the invention.

[0168]FIG. 5 shows embodiments of isolation devices with an upper partaccording to the invention.

[0169]FIG. 6 shows the ethidium bromide stained gel of anelectrophoretic separation of various samples according to the processof the invention.

[0170]FIG. 7 shows another ethidium bromide stained gel of anelectrophoretic separation of various samples according to the processof the invention.

[0171]FIG. 8 shows another ethidium bromide stained gel of anelectrophoretic separation of various samples according to the processof the invention.

[0172]FIG. 9 shows the ethidium bromide stained gel of anelectrophoretic separation of various samples according to the processof the invention.

[0173]FIG. 10 shows another ethidium bromide stained gel of anelectrophoretic separation of various samples according to the processof the invention.

[0174]FIG. 11 shows another ethidium bromide stained gel of anelectrophoretic separation of various samples according to the processof the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0175] The processes according to the invention are preferably performedin an automatic manner either partially or completely, in other words,in all stages. An example for suitable automatic equipment isillustrated in FIG. 1, in which a main part 1 is equipped with controlelectronics and driving engines with a work platform 3 and a movable arm2. Various elements are positioned on the work platform, such as area 4to hold various devices. A vacuum manifold 5 serves to absorb liquidsfrom isolation devices which are placed above it and are open at thebottom, or otherwise with the devices connected to the vacuum manifold.A shaker 6 is also provided, which can be used, e.g., for the lysis ofbiological samples. The isolation device assemblies used are, e.g.,injection-molded parts with integrated isolation devices, in which thesurfaces according to the invention are included. Typically 8, 12, 24,48, 96 or up to 1536 isolation devices can be used as these areavailable for example in the formats of modern multi-well-plates. Evenhigher numbers of isolation devices might be possible in one plate, ifstandards are available. With the aid of Luer-adapters it is, however,also possible to make separate bottoms of the assembly available and toequip these with one or more isolation devices as needed. Isolationdevices used individually without Luer-adapters are also included in theinvention.

[0176] Under a vacuum and dispensing mechanism 8 the isolation devicesare placed in the automatic apparatus and with these, liquids can betaken up and dispensed. In this assembly several single vacuum units maybe provided, so as to make the simultaneous processing of an isolationor reaction device possible. The vacuum and dispensing mechanism 8therefore acts as a pipet. Vacuum and pressure are fed to the vacuum anddispensing mechanism 8 via tube 9.

[0177] To isolate the nucleic acids, reaction devices with cells may forexample be placed in the shaker/holder 6, into which lysis buffers areintroduced with the help of the dispensing mechanism. After mixing, thecell lysates are transferred to isolation devices. The lysis buffer issubsequently passed through the surfaces in the isolation devices.Subsequently, the surfaces may be washed with a washing buffer in orderto remove cell lysate residues, in which also the washing buffer isdrained off downward. Finally, an elution buffer is dispensed into theisolation devices and after repeated shaking the separated nucleic acidsare removed from above and transferred to collection microtubes.

[0178] Usually, disposable tips are used on the vacuum and dispensingmechanism 8 to prevent contamination of the samples.

[0179]FIGS. 2 through 4 show different schematic examples for suitableisolation devices to be used according to the present invention.

[0180] In FIG. 2, a funnel-shaped isolation device 10 is provided with asurface 11, e.g., a membrane, which is placed on a collector 12, whichcontains a sponge-like material 13 that serves to absorb the lysis andwashing buffers. Under the sponge-like material 13 a superabsorbentlayer 14 may be placed to improve the suction performance.Alternatively, layer 14 may also contain a material which is chemicallyable to react with water, e.g., acrylate. The water is therefore alsoremoved from the process. Lysate or another preparation of nucleic acidsis placed in the funnel. The sponge-like material 13 absorbs the appliedliquid through membrane 11. Prior to the addition of the elution buffer,the sponge is moved some distance from the membrane, e.g., by amechanism inside a collector 12 (not visible in the drawing). This willprevent the elution buffer in the last stage from being also suctionedthrough membrane 11. This buffer, however, stays on the surface (FIG.2b) and can be removed together with the nucleic acids from above. Whenusing this assembly, the vacuum mechanism 5 in the automatic apparatusis no longer necessary.

[0181]FIG. 3 shows another example of an isolation device, which isconnected to a collector 16 via a Luer-connection located at the bottomvia a Luer-adapter 17, which in this case does not contain a sponge, butis connected to a vacuum mechanism via a muff 18. Lysis and washingbuffers may in this case be suctioned through membrane 11 by creating avacuum (FIG. 3a). When the eluate buffer is introduced, the vacuumremains turned off, so that the eluate can be removed from above (FIG.3b). With the use of a Luer-connection, individual isolation devices canbe removed from the isolation device assembly. It will be understood,however, that the vacuum collector can also be combined with fixedisolation devices, e.g., multi-well devices containing 8, 12, 24, 48, 96or more single devices.

[0182]FIG. 4 finally shows an embodiment which provides a collector,into which the buffers are suctioned through the membrane or surface 11by way of gravity or centrifuged. The eluate buffer, which is used insmall volumes, is not heavy enough itself to penetrate membrane 11 andcan again be removed from above (FIG. 4b).

[0183]FIG. 5 shows embodiments of the isolation devices according to theinvention.

[0184] In FIG. 5A, an isolation device with a cylindrical upper part 20has been illustrated. This upper part is connected to a bottom part 22by way of a threaded connection 25. Instead of the threaded connectionother types of connections may also be used, to the extent these permita watertight connection of the upper and bottom parts and provide apossibility of introducing membrane 11. In this embodiment, membrane 11is applied directly to the bottom opening of upper part 20. It may,however, also be moved inward or be placed at an angle other than 90°with respect to the upper part's wall. The bottom part also has acylindrical shape, but may be of a different design in otherembodiments. For example, a quadrangular shape may be used, whichimproves the stability of the upper part 20 on a surface. The wideningof bottom part 22 compared to upper part 20 is also possible, forexample in case a larger cavity is required in bottom part 22 in certainembodiments of the process according to the invention in order to fullyabsorb the solutions used in the absorbent material 13.

[0185] An alternative embodiment to the embodiment shown in FIG. 5A isillustrated in FIG. 5B. In this case upper part 20 and bottom part 22are fixed to one another or may also be built in one piece. Between theabsorbing material 13 and membrane 11, a sliding mechanism 27 may beslid via an opening 26 into the isolation device to separate membrane 11and absorbent material 13 from one another. In this example slidingmechanism 27 is equipped with an additional handle 28, which facilitatespulling out sliding mechanism 27. The sliding mechanism can, however,also be designed without this handle. As shown in FIG. 5B, the absorbentmaterial 13 expands slightly, to be able to bridge the space taken up bythe sliding mechanism and to make contact with the membrane.

[0186]FIG. 5C shows another embodiment of the isolation device accordingto the invention. In this case the bottom part 23 is equipped withseveral connections 30 to accommodate the upper parts 20, thuspermitting the simultaneous processing of a multiplicity of samples. Theupper parts 20 in this example are connected with bottom part 23 by wayof threaded connections 31. Although shown smaller in the illustrationthan the upper parts 20 of FIGS. 5A and 5B, it is understood that theupper parts can be the same size (or can be larger or smaller) asindicated in those embodiments.

[0187] Finally, FIG. 5D shows an isolation device according to theinvention with a collar 32 with coolant, which surrounds membrane 11 onthe outside. In this embodiment, upper part 20 and bottom part 24 areconnected to one another by way of a plug-in socket. Another type ofconnection or a one-piece version are, however, also possible. Collar 32consists of two compartments, 33 and 34, which can be connected with oneanother by destroying the separating wall 35. Both compartments 33, 34are loaded with substances, e.g. solutions, which, when mixed afterdestruction of the separating wall 35, causes the temperature of theentire mixture to drop.

[0188] The invention described above will be further explained in thefollowing examples. Different and alternative designs of the devices andprocesses will become clear to the skilled practitioner from thedescription above and from the following examples. It should expresslybe pointed out, however, that these examples and the descriptionaccompanying these examples only serve as an illustration of theinvention and are not to be considered a limitation of the invention.

EXAMPLE 1 Isolation of Total RNA from HeLa Cells

[0189] Commercially available nylon membranes (for example, a materialfrom MSI, “Magna SH” with a pore diameter of 1.2 μm, or a material fromPall GmbH, “Hydrolon” with a pore diameter of 1.2 μm), which arechemically post-treated and to be hydrophobic, were placed as a singlelayer in a plastic column. The membranes were placed on a polypropylenegrid which served as a mechanical support. The membranes were fixed inthe plastic column with a ring. The column prepared in this manner wasconnected by means of a Luer connection to a vacuum chamber. All theisolation steps were carried out through the application of a vacuum.

[0190] For the isolation, 5×10⁵ HeLa cells were harvested bycentrifugation and the supematant removed. The cells were lysed by theaddition of 150 μl; of a commercial guanidium isothiocyanate buffer(e.g., RLT buffer from QIAGEN GmbH, Hilden, Del.), in a mannerthoroughly familiar to those skilled in the art. Lysis was promoted byroughly mixing by pipetting or vortexing for 5 seconds. Then 150 μl of70% ethanol were added and mixed in by repeatedly pipetting or byvortexing for about 5 seconds.

[0191] The lysate was transferred into the plastic column and suctionedthrough the membrane by evacuating the vacuum chamber. Under theseconditions, the RNA remained bound to the membrane. Next, washing wasperformed using a first commercial washing buffer containing guanidiumisothiocyanate (e.g., with RW1 buffer from QIAGEN GmbH) and, after that,with a second washing buffer containing TRIS or TRIS and alcohol (e.g.,with the RPE buffer from QIAGEN GmbH). The washing buffers in each casewere suctioned through the membrane by evacuation of the vacuum chamber.After the final washing step, the vacuum was maintained for a period ofabout 10 minutes, in order to dry the membrane, after which the vacuumwas switched off.

[0192] For the elution, 70 μl RNase-free water was pipetted onto themembrane in order to dissolve the purified RNA from the membrane. Afterincubation for one minute at a temperature in the range from 1° to 3°C., the eluate was pipetted from the membrane from above and the elutionstep was repeated in order to make sure that the elution was complete.

[0193] The quantity of isolated total RNA obtained in this manner wasdetermined by spectrophotometric measurement of the light absorption at260 nm. The ratio between the absorbance values at 260 and 280 nm givesan estimate of RNA purity.

[0194] The results of the two isolations with hydrophobic nylonmembranes (Nos. 1 and 2) are shown in Table 1, compared with experimentsin which on the one hand a hydrophilic nylon membrane (Nyaflo) (No. 3)and a silica membrane (No. 4) were used. The values reported in thetable provide convincing support for the impressive isolation yield andseparation effect of the materials used in accordance with thisinvention. They also show that silica gel-fleece clearly produces alower yield, which presumably can be attributed to its fleecelikestructure and the ensuing absorption of a large portion of the eluatebuffer. TABLE 1 RNA yield and purity of total RNA isolated according toExample 1. Yield of Total-RNA Absorbance Sample No. Type of Membrane(μg) E₂₆₀/E₂₈₀ 1 Magna SH 1.2 μm 6.0 1.97 (hydrophobic nylon) 2 Hydrolon1.2 μm 7.1 2.05 (hydrophobic nylon) 3 Nylaflo (hydrophilic <0.2 Notnylon) Determined 4 hydrophilic silica <0.2 Not membrane Determined

[0195] The isolated RNA can also be analyzed on agarose gels that havebeen stained with ethidium bromide. For this purpose, for example, 1.2%formaldehyde agarose gels were prepared. The result is shown in FIG. 6.In FIG. 6, Lane 1 is the total RNA that was isolated on a hydrophobicnylon membrane (Magna SH, Sample no. 1) with a pore diameter of 1.2 μm.Lane 2 is total RNA that was isolated by means of a hydrophobic nylonmembrane (Hydrolon, Sample no. 2) with a pore diameter of 1.2 μm. Lane 3represents the chromatogram of a total RNA that was isolated by means ofa silica membrane (Sample no. 4). In each case, 50 μl of the total RNAeluate was analyzed. FIG. 6 provides convincing evidence that when asilica membrane was used, no measurable proportion of the total RNA canbe isolated.

EXAMPLE 2 Isolation of Free RNA by Binding the RNA to HydrophobicMembranes by Means of Various Salt-alcohol Mixtures

[0196] In this example, the lysate and washing solutions are conductedthrough the hydrophobic membrane by applying a vacuum.

[0197] Hydrophobic nylon membranes (e.g., 1.2 μm Hydrolon from Pall)were introduced into plastic columns connected to a vacuum chamber, in amanner similar to that of Example 1. To 100 μl aliquots of an aqueoussolution containing total RNA were added 350 μl of a commerciallyavailable lysis buffer containing guanidium isothiocyanate (e.g., RLTbuffer from QIAGEN), 350 μl of 1.2 M sodium acetate solution, or 350 μlof 4 M lithium chloride solution, respectively, and the resultingsolutions were mixed by pipetting.

[0198] Next, 250 μl of ethanol were added to each mixture and mixed,likewise by pipetting. After that, the solutions containing RNA weretransferred into the plastic columns and suctioned through the membraneby evacuating the vacuum chamber. Under the conditions described, theRNA remains bound to the membranes. The membranes were then washed, asdescribed in Example 1. Finally, the RNA, also as described in Example1, was removed from the membrane by pipetting from above.

[0199] The quantity of isolated total RNA was determined byspectrophotometric measurement of the light absorption at 260 nm. Theratio between the absorbance values at 260 and 280 nm gives an estimateof RNA purity. The results are set forth in Table 2 below. TABLE 2Isolation of RNA from aqueous solution by binding the RNA to hydrophobicmembranes using various salt-alcohol mixtures. Yield of Total Sample RNAAbsorbance No. Salt/Alcohol mixture (μg) E₂₆₀/E₂₈₀ 1 RLT-BufferQIAGEN/35% Ethanol 9.5 1.92 2 0.6 M Sodium Acetate/35% Ethanol 8.5 1.983   1 M Sodium Chloride/35% Ethanol 7.9 1.90 4   2 M LithiumChloride/35% Ethanol 4.0 2.01

EXAMPLE 3 Isolation of Total RNA from HeLa Cells

[0200] Following the procedures of Example 1, plastic columns wereassembled with different hydrophobic membranes. Each column thusprepared was placed in a collection tube, and the following isolationsteps were performed by way of centrifugation.

[0201] For the isolation, 5×10⁵ HeLa cells were harvested bycentrifugation and the supernatant removed. The cells were lysed by theaddition of 150 μl of a commercially available guanidiniumisothiocyanate buffer, such as, e.g., RLT-buffer from QIAGEN, using wellknown procedures. In this connection, lysis is encouraged by multiplepipetting or by vortexing for 5 seconds. Subsequently, 150 μl of 70%ethanol was added and mixed by multiple pipetting or by vortexing for 5seconds.

[0202] The lysate was subsequently transferred into a plastic column andpassed through the membrane by centrifugation at 10000×g for 1 minute.Subsequently, washing was performed with a commercially availablewashing buffer containing guanidinium isothiocyanate, e.g., with theRW1-buffer of QIAGEN, followed by a second washing step using a buffercontaining TRIS and alcohol, e.g., RPE-buffer from QIAGEN. The washingbuffers were passed through the membrane by centrifugation. The lastwashing step takes place at 20000×g for 2 minutes to dry the membrane.

[0203] For elution, 70 μl of RNase-free water were pipetted onto themembrane to release the purified RNA from the membrane. After a 1-2minute incubation at a temperature between 10°-30° C., the eluate wastaken from above by pipetting from the membrane. The elution step wasrepeated once to achieve complete elution.

[0204] The quantity of isolated total RNA was determined byspectrophotometric measurement of the light absorption at a wavelengthof 260 nm. RNA quality is determined by spectrophotometic determinationof the light absorption ratio compared at 260 nm and at 280 nm. Theisolation results with different hydrophobic membranes are listed inTable 3 below. The data represent the average of 3-5 parallel tests permembrane. Using a silica membrane, no measurable quantity of total RNAcould be isolated, where the eluate was recovered by removing it fromabove from the membrane. TABLE 3 Yield of total RNA isolated by bindingto hydrophobic membranes. Manufacturer Membrane Material RNA(μg)E₂₆₀/E₂₈₀ Pall Hydrolon, 1.2 μm Hydrophobic Nylon 6.53 1.7 PallHydrolon, 3 μm Hydrophobic Nylon 9.79 1.72 Pall Fluoro Trans GHydrophobic Polyvinylidene 6.16 1.72 Fluoride Pall Fluoro Trans WHydrophobic Polyvinylidene 5.4 1.9 Fluoride Pall Bio Trace HydrophobicPolyvinylidene 4.3 1.97 Fluoride Pall Supor-450 PR HydrophobicPolyethersulfone 3.96 1.76 Pall V-800 R Hydrophobic Acryliccopolymer6.26 1.72 Pall Versapor - 1200 R Hydrophobic Acryliccopolymer 6.23 1.68Pall Versapor - 3000 R Hydrophobic Acryliccopolymer 3.54 1.74 Gore-TexOH 9335 Hydrophobic Poly-Tetrafluoroethylene 1.59 1.72 Gore-Tex OH 9336Hydrophobic Poly-Tetrafluoroethylene 2.15 1.65 Gore-Tex OH 9337Hydrophobic Poly-Tetrafluoroethylene 3.6 1.59 Gore-Tex QH 9316Hydrophobic Poly-Tetrafluoroethylene 3.61 1.69 Gore-Tex QH 9317Hydrophobic Poly-Tetrafluoroethylene 2.87 1.70 Millipore Mitex MembraneHydrophobic Poly-Tetrafluoroethylene 1.98 1.62 Millipore DuraporeHydrophobic Polyviylidene 7.45 1.72 Fluoride MSI Magna-SH, 1.2 μmHydrophobic Nylon 4.92 1.69 MSI Magna-SH, 5 μm Hydrophobic Nylon 10.21.71 MSI Magna-SH, 10 μm Hydrophobic Nylon 7.36 1.76 MSI Magna-SH, 20 μmHydrophobic Nylon 7.04 1.65 Sartorius Type 118 HydrophobicPoly-Tetrafluoroethylene 7.6 1.61 Mupor PM12A HydrophobicPoly-Tetrafluoroethylene 6.7 1.77 Mupor PM3VL HydrophobicPoly-Tetrafluoroethylene 6.6 1.77

EXAMPLE 3b Isolation of Total RNA from HeLa-cells by Binding toHydrophilic Membranes

[0205] Using the procedures of Example 1, plastic columns were assembledusing different hydrophilic membranes. Each column thus prepared wasplaced in a collection tube, and the following isolation steps wereperformed by centrifugation.

[0206] For the isolation, 5×10⁵ HeLa cells were used. The isolationsteps and elution of the nucleic acids were carried out as describedabove in Example 3 for hydrophobic membrane columns.

[0207] The quantity of isolated total RNA was determined byspectrophotometric measurement of the light absorption at a wavelengthof 260 nm. RNA quality was determined by the spectrophotometricdetermination of the ratio of the light absorption compared at 260 nmand at 280 nm. The isolation results with various hydrophilic membranesare listed in Table 3b below. The data represent the average of 2-5parallel tests per membrane. Using a silica membrane, no measurablequantity of total RNA could be isolated, where the eluate was recoveredby removing it from above from the membrane. TABLE 3b Yield of total RNAisolated by binding to hydrophilic membranes. Manufacturer MembraneMaterial RNA (μg) E₂₆₀/E₂₈₀ Pall Loprodyne Hydrophilic Nylon 3.1 1.8Pall Loprodyne Hydrophilic Nylon 3.1 1.78 Pall Biodyne A HydrophilicNylon 3.1 1.8 Pall Biodyne A Hydrophilic Nylon 3.6 1.83 Pall Biodyne BHydrophilic Nylon 2.6 1.84 Pall Biodyne B Hydrophilic Nylon 4.2 1.84Pall Biodyne C Hydrophilic Nylon 6.1 1.88 Pall Biodyne C HydrophilicNylon 5.2 1.91 Pall Biodyne plus Hydrophilic Nylon 3.3 1.87 PallI.C.E.-450 Hydrophilic Polyethersulfone 6.36 1.8 Pall I.C.E.-450 supHydrophilic Polyethersulfone 3.07 1.71 Pall Supor - 800 HydrophilicPolyethersulfone 4.12 1.7 Pall Supor - 450 Hydrophilic Polyethersulfone4.69 1.69 Pall Supor - 100 Hydrophilic Polyethersulfone 3.25 1.71 PallHemasep V Hydrophilic Polyester 4.16 1.74 Pall Hemasep L HydrophilicPolyester 6.67 1.65 Pall Leukosorb Hydrophilic Polyester 1.5 1.84 PallPremium Release Hydrophilic Polyester 1.66 1.63 Membrane PallPolypro-450 Hydrophilic Polypropylene 5.09 1.78 Gore-Tex OH 9339Hydrophilic Poly-Tetrafluoroethylene 1.08 1.65 Gore-Tex OH 9338Hydrophilic Poly-Tetrafluoroethylene 3.97 1.67 Gore-Tex QH 9318Hydrophilic Poly-Tetrafluoroethylene 3.61 1.69 Millipore DuraporePolyvinylidene Fluoride made 5.6 1.69 Hydrophilic Millipore DuraporePolvinylidene Fluoride made 3.12 1.68 Hydrophilic Millipore LCRPoly-Tetrafluoroethylene 3.14 1.66 made Hydrophilic Sartorius Type 250Hydrophilic Polyamide 4.3 1.66 Sartorius Type 113 Hydrophilic CelluloseNitrate 1.8 1.86 Sartorius Type 113 Hydrophilic Cellulose Nitrate 1.91.74 Infiltec Polycone, 0.01 Hydrophilic Polycarbonate 0.17 1.64Infiltec Polycone, 0.1 Hydrophilic Polycarbonate 0.73 1.68 InfiltecPolycone, 1 Hydrophilic Polycarbonate 3.33 1.86

EXAMPLE 4 Isolation of Free RNA from an Aqueous Solution

[0208] Using the procedures according to Example 1, plastic columns wereassembled with different hydrophobic membranes. 100 μl of an aqueoussolution containing total RNA were mixed with 350 μl of a commerciallyavailable lysis buffer containing guanidinium-isothiocyanate, e.g.,RLT-buffer from QIAGEN. Subsequently, 250 μl of ethanol were added andmixed by pipetting. This mixture was then introduced to the column andpassed through by centrifugation (10000×g; 1 minute) through themembrane. The membranes were subsequently washed twice with a washingbuffer, e.g., RPE from QIAGEN. The buffer was passed through themembranes by centrifugation. The last washing step was carried out at20000×g to dry the membranes.

[0209] Next, the RNA, as described in Example 1, was eluted withRNase-free water and removed from the membrane from above by pipetting.The quantity of isolated total RNA was determined by spectrophotometricmeasurement of light absorption at a wavelength of 260 nm. RNA qualitywas determined by the spectrophotometric determination of the ratio ofthe light absorption at 260 nm to 280 nm. The isolation results withvarious hydrophobic membranes are listed in Table 4 below. The datarepresent the average of 3-5 parallel tests per membrane. Using a silicamembrane, no measurable quantity of total RNA could be isolated, wherethe eluate was recovered by removing it from above from the membrane.TABLE 4 Isolation of free RNA from an aqueous solution by binding tohydrophobic membranes. Manufacturer Membrane Material RNA(μg) E₂₆₀/E₂₈₀Pall Hydrolon, 1.2 μm Hydrophobic Nylon 5.15 1.75 Pall Hydrolon, 3 μmHydrophobic Nylon 0.22 1.79 Pall Fluoro Trans G HydrophobicPolyvinylidene Fluoride 5.83 1.79 Pall Fluoro Trans W HydrophobicPolyvinylidene Fluoride 5.4 1.84 Pall Bio Trace HydrophobicPolyvinylidene Fluoride 4.0 1.79 Pall Emflon HydrophobicPoly-Tetrafluor-Ethylene 0.2 1.7 Pall Supor-450 PR HydrophobicPolyethersulfone 5.97 1.71 Pall Supor-200 PR HydrophobicPolyethersulfone 2.83 1.66 Pall V-800 R Hydrophobic Acrylatecopolymer2.74 1.77 Gore-Tex OH 9335 Hydrophobic Poly-Tetrafluor-Ethylene 4.351.63 Gore-Tex OH 9336 Hydrophobic Poly-Tetrafluor-Ethylene 7.43 1.71Gore-Tex OH 9337 Hydrophobic Poly-Tetrafluor-Ethylene 5.96 1.62 Gore-TexQH 9316 Hydrophobic Poly-Tetrafluor-Ethylene 5.92 1.67 Gore-Tex QH 9317Hydrophobic Poly-Tetrafluor-Ethylene 8.7 1.66 Millipore FluoroporeHydrophobic Poly-Tetrafluor-Ethylene 8.46 1.70 Millipore Durapore, 0.65μm Hydrophobic Polyvinylidene Fluoride 4.23 1.8 MSI Magna-SH, 1.2 μmHydrophobic Nylon 1.82 1.76 MSI Magna-SH, 5 μm Hydrophobic Nylon 0.61.78 Sartorius Type 118 Hydrophobic Poly-Tetrafluor-Ethylene 0.9 1.82Sartorius Type 118 Hydrophobic Poly-Tetrafluor-Ethylene 5.4 1.74 MuporPM12A Hydrophobic Poly-Tetrafluor-Ethylene 1.1 1.98

EXAMPLE 4b Isolation of Free RNA from an Aqueous Solution by Binding toHydrophilic Membranes

[0210] Following the procedures of Example 1, plastic columns wereassembled using different hydrophilic membranes.

[0211] 100 μl of an aqueous solution containing total RNA were mixedwith 350 μl of a commercially available lysis buffer containingguanidinium-isothiocyanate, e.g., RLT-buffer from QIAGEN. Subsequently250 μl of ethanol were added and mixed by pipetting back and forth. Thismixture was then introduced to the column, passed through the membrane,washed and dried according to the procedure used in Example 4, above.

[0212] Finally, the RNA, as described in Example 1, was eluted withRNase-free water and removed from the membrane using a pipette.

[0213] The quantity of isolated total RNA was determined byspectrophotometric measurement of the light absorption at a wavelengthof 260 nm. RNA quality was determined by the spectrophotometricdetermination of the ratio of the light absorption compared at 260 nmand at 280 nm. The isolation results with various hydrophilic membranesare listed in Table 4b below. The data represent the average from 2-5parallel tests per membrane. Using a silica membrane, no measurablequantity of total RNA could be isolated, where the eluate was recoveredby removing it from above from the membrane. TABLE 4b Isolation of freeRNA from an aqueous solution by binding to hydrophilic membranes.Manufacturer Membrane Material RNA (μg) E₂₆₀/E₂₈₀ Pall LoprodyneHydrophilic Nylon 2 1.8 Pall Loprodyne Hydrophilic Nylon 1.4 1.87 PallBiodyne A Hydrophilic Nylon 4.5 1.93 Pall Biodyne A Hydrophilic Nylon3.1 1.9 Pall Biodyne B Hydrophilic Nylon 1.7 1.94 Pall Biodyne BHydrophilic Nylon 1.2 1.94 Pall Biodyne C Hydrophilic Nylon 3.7 1.93Pall Biodyne C Hydrophilic Nylon 3.1 1.93 Pall Biodyne plus HydrophilicNylon 1.1 1.87 Pall I.C.E.-450 Hydrophilic Polyethersulfone 1.92 1.82Pall I.C.E.-450 sup Hydrophilic Polyethersulfone 0.87 1.67 Pall Supor -800 Hydrophilic Polyethersulfone 3.93 1.74 Pall Supor - 450 HydrophilicPolyethersulfone 1.78 1.74 Pall Supor - 100 Hydrophilic Polyethersulfone1.04 1.68 Pall Hemasep V Hydrophilic Polyester 4 1.79 Pall Hemasep LHydrophilic Polyester 0.47 2.1 Pall Polypro - 450 HydrophilicPolypropylene 5.09 1.78 Gore-Tex OH 9339 HydrophilicPoly-Tetrafluor-Ethylene 0.43 1.48 Gore-Tex OH 9338 HydrophilicPoly-Tetrafluor-Ethylene 3.63 1.64 Gore-Tex QH 9318 HydrophilicPoly-Tetrafluor-Ethylene 5.92 1.67 Millipore Durapore PolyvinylideneFluoride made 1.18 1.79 Hydrophilic Millipore LCRPoly-Tetrafluor-Ethylene made 2.84 1.72 Hydrophilic Sartorius Type 250Hydrophilic Polyamide 2.7 1.7 Sartorius Type 111 Hydrophilic CelluloseAcetate 1.6 1.85 Sartorius Type 111 Hydrophilic Cellulose Acetate 2.22.1 Sartorius Type 111 Hydrophilic Cellulose Acetate 0.3 2.01 SartoriusType 113 Hydrophilic Cellulose Nitrate 4 1.88 Sartorius Type 113Hydrophilic Cellulose Nitrate 3.8 1.87

EXAMPLE 5 Isolation of Total RNA from HeLa-cells Depending on the PoreSize of the Membranes

[0214] Following the procedures of Example 1, plastic columns wereassembled with different hydrophobic membranes with different poresizes.

[0215] As in Example 3, a cell lysate was made from 5×10⁵ HeLa cells andtransferred to the columns. Subsequently the membranes were washed withthe commercially available buffers RW1 and RPE from QIAGEN. The lastcentrifugation step was carried out at 20000×g for 2 minutes to dry themembrane. The elution was carried out as described in Example 1.

[0216] The results are listed in Table 5 below. 3-5 parallel tests permembrane were performed and the average value calculated for each. TABLE5 Yield of isolated total RNA using hydrophobic membranes with differentpore sizes. Pore Size RNA Manufacturer Membrane Material (μm) (μg)E₂₆₀/E₂₈₀ Infiltec Polycon 0.01 Hydrophilic Polycarbonate 0.01 0.17 1.64Pall Fluoro Trans G Hydrophobic 0.2 6.16 1.72 Polyvinylidene FluoridePall Supor-450 PR Hydrophobic 0.45 3.96 1.76 Polyethersulfone MilliporeDurapore Hydrophobic 0.65 7.45 1.72 Polyvinylidene Fluoride MSI Magna-SHHydrophobic Nylon 1.2 4.92 1.69 MSI Magna-SH Hydrophobic Nylon 5 10.21.71 MSI Magna-SH Hydrophobic Nylon 10 7.36 1.76 MSI Magna-SHHydrophobic Nylon 20 7.04 1.65

EXAMPLE 6 Stability and Quality of Isolated Total RNA from HeLa Cells

[0217] According to procedures of Example 1, plastic columns wereassembled with a commercially available membrane (Pall, Hydrolon with a3 μm pore size).

[0218] According to the procedures of Example 3, a cell lysate was madefrom 5×10⁵ HeLa cells and transferred to the columns. Subsequently, themembranes were washed with the commercially available buffers RW1 andRPE from QIAGEN. The last centrifugation step was carried out at 20000×gfor 2 minutes to dry the membrane. The elution was carried out asdescribed in Example 1.

[0219] The isolated total RNA was left to incubate for 16 hours at 37°C. and subsequently placed on a denaturating agarose gel and analyzed.It was demonstrated that the RNA did not suffer degradation. The RNAisolated with the method described above shows no contaminants withenzymes that degrade nucleic acids and therefore is of high quality.

EXAMPLE 7 Isolation of Free RNA from an Aqueous Solution by Binding to aHydrophilic Membrane in a 96-well Plate

[0220] A 96-well plate with a hydrophilic Polyvinylidene Fluoridemembrane (Durapore, 0.65 μm by Millipore) was used. 5.3 ml of an aqueoussolution containing total RNA were mixed with 18.4 ml of a commerciallyavailable lysis buffer containing guanidinium isothiocyanate, e.g., RLTbuffer from QIAGEN. Subsequently 13.1 ml ethanol were added and mixed bypipetting back and forth. For each well, 350 μl of this mixture wereintroduced and passed through the membrane by applying a vacuum. Themembranes were subsequently washed twice with a buffer, e.g., RPE fromQIAGEN. The buffer was passed through the membrane each time by applyinga vacuum. After the last washing step, the plate was dabbed once with apaper towel and subsequently dried for 5 minutes by applying a vacuum.

[0221] The RNA was eluted as described in Example 1, with RNase-freewater and removed from the membrane by way of a pipette. The quantity ofisolated total RNA was determined by spectrophotometric measurement ofthe light absorption at a wavelength of 260 nm and the average value aswell as the standard deviation for the entire plate was calculated. Theaverage value is 8.4 μg with a standard deviation of 0.7 μg.

EXAMPLE 8 Isolation of Total RNA by Way of Capillary Forces

[0222] A 96-well plate with a hydrophilic Polyvinylidene Fluoridemembrane (Durapore, 0.65 μm by Millipore) was used. 33 μl of an aqueoussolution containing total RNA were mixed with 110 μl of a commerciallyavailable lysis buffer containing guanidinium isothiocyanate, e.g., RLTbuffer from QLAGEN. Subsequently 78 μl ethanol were added and mixed bypipetting. 45 μl of this mixture were introduced into each well. Anabsorbent household sponge was moistened with water, and the 96-wellplate was placed with the membrane's bottom side on the sponge. The RNAmixture was passed through the membrane by way of capillary forces. Themembranes were subsequently washed twice with a buffer, e.g., RPE fromQIAGEN. The wash buffer was also passed through the membrane by placingthe plate on the sponge. After the last washing step, the plate wasair-dried for 5 minutes.

[0223] The RNA, as described in Example 1, was eluted with RNase-freewater and removed from the membrane by way of a pipette.

[0224] The quantity of isolated total RNA is subsequently determined byspectrophotometric measurement of the light absorption at a wavelengthof 260 nm, and the average value as well as the standard deviation iscalculated. The average value is 5.9 μg with a standard deviation of 0.7μg.

EXAMPLE 9 Isolation of Genomic DNA from an Aqueous Solution by Way of aBuffer Containing Guanidinium Hydrochloride

[0225] According to Example 1, plastic columns were assembled withhydrophobic membranes (e.g., Magna-SH, 5 μm by the MSI Company).Purification is carried out with commercially available buffers fromQIAGEN.

[0226] 200 μl of an aqueous solution of genomic DNA from liver tissuewere introduced in PBS buffers. 200 μl of a buffer containingguanidinium hydrochloride, e.g. QIAGEN's AL, were added to and mixedwith this solution. Subsequently 210 μl of ethanol were added and mixedthrough vortexing. The mixture was introduced to the column according toExample 3 and passed through the membrane by way of centrifugation. Themembrane was then washed and dried with an alcohol containing buffer,e.g., QIAGEN's AW. The elution was performed as described in Example 1.Three parallel tests were carried out and the average value calculated.The amount of isolated DNA is subsequently determined byspectrophotometric measurement of the light absorption at a wavelengthof 260 nm and is approx. 30% of the starting amount. The absorptionratio at 260 nm to 280 nm is 1.82.

EXAMPLE 10 Isolation of Genomic DNA from an Aqueous Solution by Bindingto Hydrophobic Membranes by Way of a Buffer Containing GuanidiniumIsothiocyanate

[0227] According to Example 1, plastic columns were assembled withdifferent membranes. 100 μl of an aqueous solution containing total DNAwere mixed with 350 μl of a lysis buffer containing guanidiniumisothiocyanate (4 M GITC, 0.1 M MgSO₄, 25 mM Na-Citrate, pH 4).Subsequently 250 μl ethanol were added and mixed by pipetting. Thismixture was then transferred to the column and passed through themembrane by way of centrifugation (10000×g; 1 minute). The membraneswere subsequently washed twice with a buffer, e.g., RPE by QIAGEN. Thebuffer was passed through the membranes by way of centrifugation. Thelast washing step was carried out at 20000×g to dry the membranes.

[0228] The elution was performed as described in Example 1. Threeparallel tests were carried out per membrane and the average value iscalculated each time. The results are listed in Table 6. TABLE 6DNA-yield from an aqueous solution by binding to hydrophobic membranesManufacturer Membrane Material DNA (μg) Pall Hydrolon, 1.2 μmHydrophobic Nylon 1.3 Pall Supor-450 PR Hydrophobic 2.2 PolyethersulfonMillipore Fluoropore Hydrophobic 1.1 Poly-Tetrafluor-Ethylene MilliporeDurapore Hydrophobic 1.2 Polyvinylidene Fluoride

EXAMPLE 11 Isolation of Genomic DNA from Tissue

[0229] According to Example 1, plastic columns were assembled withhydrophobic membranes (e.g., Magna-SH, 5 μm by MSI). Purification wascarried out with the commercially available buffers from QIAGEN.

[0230] 180 μl of ATL-buffer were added to 10 mg of kidney tissue (mouse)and ground in a mechanical homogenizer. Subsequently proteinase K(approx. 0.4 mg dissolved in 20 μl of water) were added and incubatedfor 10 minutes at 55° C. After adding 200 μl of a buffer containingguanidinium hydrochloride, e.g., AL by QIAGEN, and after a 10-minuteincubation at 70° C., 200 μl of ethanol were added and mixed with thissolution. This mixture was transferred on to the column and passedthrough the membrane by centrifugation. The membrane was then washedwith alcohol containing buffers, e.g., AW1 and AW2 from QIAGEN, andsubsequently dried by way of centrifugation. The elution was carried outas described in Example 1. Three parallel tests were carried out and theaverage value calculated.

[0231] The amount of isolated DNA, determined by spectrophotometricmeasurement of the light absorption at a wavelength of 260 nm, was onaverage 9.77 μg. The absorption ratio at 260 nm to 280 nm was 1.74.

EXAMPLE 12 Isolation of Genomic DNA from Blood

[0232] According to the procedures of Example 1, plastic columns wereassembled with hydrophobic membranes (e.g., Magna-SH, 5 μm by MSI).Purification was carried out with the commercially available buffersfrom QIAGEN.

[0233] 200 μl of AL buffer and 20 μl of QIAGEN protease were added to200 μl of blood, thoroughly mixed, and left to incubate for 10 minutesat 56° C. After adding 200 μl of ethanol, the solution was mixed,transferred onto the column, and passed through the membrane by way ofcentrifugation. The membrane was then washed with alcohol containingbuffers, e.g., AW1 and AW2 from QIAGEN, and subsequently dried by way ofcentrifugation. The elution was carried out as described in Example 1.

[0234] The amount of isolated DNA, determined by spectrophotometricmeasurement of the light absorption at a wavelength of 260 nm, was 1.03μg. The absorption ratio at 260 nm 280 nm is 1.7.

EXAMPLE 13 Isolation of Total RNA from an RNA-DNA-mixture

[0235] Following the procedures of Example 1, plastic columns wereassembled with hydrophobic membranes (e.g., Hydrolon 1.2 μm by the PallCompany). 275 μl of an aqueous solution containing total RNA and genomicDNA were mixed with 175 μl of a commercially available lysis buffercontaining guanidinium isothiocyanate, e.g., the RLT buffer from QIAGEN.250 μl of ethanol were added and mixed by pipetting. The mixture wastransferred to the column and passed through the membrane, washed anddried according to Example 4. The flow-through from the firstcentrifugation step was placed on a commercially available mini-spincolumn (e.g., QIAamp Mini-Spin Column from QIAGEN) and passed throughthe membrane via centrifugation. The remaining washing steps wereperformed as described in Example 4.

[0236] After this, the nucleic acids were eluted with 140 μl ofRNase-free water by way of centrifugation (10000×g, 1 minute) andanalyzed in non-denaturing agarose gel (see FIG. 7). The major part ofthe total RNA can be separated from the genomic DNA with the use of themethod described above.

[0237]FIG. 7 shows an ethidium-bromide stained gel of an electrophoreticseparation of two different eluates.

[0238] Lane 1: Isolation of total RNA by way of a hydrophobic nylonmembrane.

[0239] Lane 2: Isolation of genomic DNA from the flow-through by way ofa QIAamp mini-spin column of the QIAGEN company.

EXAMPLE 14 Isolation of Plasmid DNA from an Aqueous Solution by Bindingto Hydrophobic and Hydrophilic Membranes

[0240] Following the procedures of Example 1, plastic columns wereassembled utilizing different membranes.

[0241] 100 μl of an aqueous solution (pCMVβ from Clontech) containingplasmid were mixed with 350 μl of lysis buffer containing guanidiniumisothiocyanate (4 M GITC, 0.1 M MgSO₄, 25 mM sodium-acetate, pH 4).Subsequently, 250 μl of isopropanol were added and mixed by pipetting.This mixture was then transferred onto one of the columns and passedthrough the membrane, washed and dried according to the proceduresdescribed in Example 4. Finally the plasmid DNA, as described previouslyin Example 1, was eluted with RNase-free water and removed from themembrane by pipetting.

[0242] The amount of isolated plasmid DNA was determined byspectrophotometric measurement of the light absorption at a wavelengthof 260 nm. The isolation results using various membranes are listed inTable 7 below. Three parallel tests per membrane were carried out andeach time the average value is calculated. TABLE 7 Plasmid DNA-yieldfrom an aqueous solution by binding to membranes Plasmid DNAManufacturer Membrane Material (μg) Pall Hydrolon, 1.2 μm HydrophobicNylon 1.9 Pall Fluoro Trans G Hydrophobic Polyvinylidene Fluoride 2.2Pall I.C.E.-450 Hydrophilic Polyethersulfone 0.8 Pall I.C.E.-450 supHydrophilic Polyethersulfone 1.5 Pall Supor-450 PR HydrophobicPolyethersulfone 4.7 Pall Supor-200 PR Hydrophobic Polyethersulfone 4Pall Supor - 800 Hydrophilic Polyethersulfone 0.5 Pall Supor - 450Hydrophilic Polyethersulfone 0.9 Pall Supor - 100 HydrophilicPolyethersulfone 1 Pall V-800 R Hydrophobic Acrylic Copolymer 1.5 PallVersapore-1200 R Hydrophobic Acrylic Copolymer 0.2 Pall Polypro-450Hydrophilic Polypropylene 1.4 Gore-Tex QH 9318 HydrophilicPoly-Tetrafluoro-Ethylene 4.9 Gore-Tex OH 9335 HydrophobicPoly-Tetrafluoro- 4.3 Ethylene Millipore Durapore, 0.65 μmPolyvinylidene Fluoride made 1.8 Hydrophobic Millipore Durapore, 0.65 μmHydrophobic Polyvinylidene Fluoride 1.7 MSI Magna-SH, 1.2 μm HydrophobicNylon 1.1

EXAMPLE 15 Immobilization of Total RNA from an Aqueous Solution with theUse of Different Chaotropic Agents

[0243] Following the procedures of Example 1, plastic columns wereassembled utilizing different hydrophobic membranes.

[0244] 100 μl of an aqueous solution containing total RNA were mixedwith 350 μl of different lysis buffers, which contain guanidiniumisothiocyanate (GITC) or guanidinium hydrochloride (GuHCl) in differentconcentrations. 250 μl ethanol were added and mixed by pipetting. Thismixture was then placed on one of the columns and passed through themembrane by way of centrifugation (10000×g; 1 minute). The membraneswere subsequently washed twice with an alcohol containing buffer, e.g.,RPE from QIAGEN. The buffer was passed through the membrane bycentrifugation. The last washing step was performed at 20000×g to drythe membrane. The elution was carried out as described in Example 1. Twotests were carried out to determine the average value. The results arelisted in Table 8. TABLE 8 RNA-yield from an aqueous solution by way ofchaotropic agents Chaotropic Agents, Concentration in Binding MembraneSolution Yield of Total RNA (μg) Hydrolon, 1.2 μm GITC, 500 mM 2.3Hydrolon, 1.2 μm GITC, 1 M 0.8 Hydrolon, 1.2 μm GITC, 3 M 0.9 FluoroTrans G GITC, 500 mM 0.4 Fluoro Trans G GITC, 1 M 1.25 Fluoro Trans GGITC, 3 M 0.6 Hydrolon, 1.2 μm GuHCI, 500 mM 2.6 Hydrolon, 1.2 μm GuHCI,1 M 6.7 Hydrolon, 1.2 μm GuHCI, 3 M 2.9 Fluoro Trans G GuHCI, 500 mM 0.4Fluoro Trans G GuHCI, 1 M 1.25 Fluoro Trans G GuHCI, 3 M 0.6

EXAMPLE 16 Immobilization of Total RNA from an Aqueous Solution UsingAlcohols

[0245] Following the procedures of Example 1, plastic columns wereassembled utilizing different hydrophobic membranes. 100 μl of anaqueous solution containing total RNA are mixed with 350 μl of a lysisbuffer containing guanidinium isothiocyanate (concentration 4 M).Different amounts of ethanol and isopropanol were added and filled withRNase-free water up to 700 μl and mixed. This mixture was thenintroduced to a column and passed through the membrane and washedaccording to the procedures of Example 4. The elution took place as inExample 1. Two tests were carried out to determine the average yield.The results are listed in Table 9. TABLE 9 RNA-yield from an aqueoussolution with different alcohols in a binding solution Alcohol,Concentration Membrane in Binding Solution Yield of Total RNA (μg)Hydrolon, 1.2 μm Ethanol, 5% 0.7 Hydrolon, 1.2 μm Ethanol, 30% 2.85Hydrolon, 1.2 μm Ethanol, 50% 4.5 Durapore, 0.65 μm Ethanol, 5% 0.4Durapore, 0.65 μm Ethanol, 30% 1.25 Durapore, 0.65 μm Ethanol, 50% 0.6Hydrolon, 1.2 μm Isopropanol, 5% 0.35 Hydrolon, 1.2 μm Isopropanol, 30%4.35 Hydrolon, 1.2 μm Isopropanol, 50% 3.2 Durapore, 0.65 μmIsopropanol, 10% 1.35 Durapore, 0.65 μm Isopropanol, 30% 4.1 Durapore,0.65 μm Isopropanol, 50% 3.5

EXAMPLE 17 Immobilization of Total RNA from an Aqueous Solution withVarious pH-values

[0246] Using the procedures described in Example 1, plastic columns wereassembled utilizing various hydrophobic membranes. 100 μl of an aqueoussolution containing total RNA were mixed with 350 μl of a lysis buffercontaining guanidinium isothiocyanate (concentration 4 M). The buffercontained 25 mM of sodium citrate and was adjusted to differentpH-values with HCl or NaOH. Subsequently, 250 μl of ethanol were addedand mixed. This mixture was then introduced to the column and passedthrough the membrane and washed according to the procedures of Example4. The elution took place as in Example 1. Two tests are carried out todetermine an average value. The results are listed in Table 10. TABLE 10RNA-yield from an aqueous solution with various pH-values in a bindingsolution Membrane pH of Binding Solution Yield of Total RNA (μg)Hydrolon, 1.2 μm pH 3 0.15 Hydrolon, 1.2 μm pH 9 1.6  Hydrolon, 1.2 μm pH 11 0.05 Fluoro Trans G pH 1 0.45 Fluoro Trans G pH 9 2.85 FluoroTrans G  pH 11 0.25

EXAMPLE 18 Immobilization of Total RNA from an Aqueous Solution withVarious Salts

[0247] According to Example 1, plastic columns are assembled withhydrophobic membranes. 100 μl of a total RNA containing aqueous solutionwere mixed with 350 μl of a salt containing lysis buffer (NaCl, KCL,MgSO₄). 250 μl of H₂O or ethanol were then added and mixed. This mixturewas then transferred to a column and passed through the membrane, washedand eluted according to the procedures of Example 4. Two tests werecarried out to determine the average value. The results are listed inTable 11. TABLE 11 RNA-yield from an aqueous solution with various saltsin the binding solution Salt Yield of Total Membrane Concentration inBinding Solution RNA (μg) Hydrolon, 1.2 μm NaCl, 100 mM; without ethanol0.1 Hydrolon, 1.2 μm NaCl, 1 M; without ethanol 0.15 Hydrolon, 1.2 μmNaCl, 5 M; without ethanol 0.3 Hydrolon, 1.2 μm KCl, 10 mM; withoutethanol 0.2 Hydrolon, 1.2 μm KCl, 1 M; without ethanol 0.1 Hydrolon, 1.2μm KCl, 3 M; without ethanol 0.25 Hydrolon, 1.2 μm MgSO₄, 100 mM;without ethanol 0.05 Hydrolon, 1.2 μm MgSO₄, 750 mM; without ethanol0.15 Hydrolon, 1.2 μm MgSO₄, 2 M; without ethanol 0.48 Hydrolon, 1.2 μmNaCl, 500 mM; with ethanol 2.1 Hydrolon, 1.2 μm NaCl, 1 M; with ethanol1.55 Hydrolon, 1.2 μm NaCl, 2.5 M; with ethanol 1.35 Hydrolon, 1.2 μmKCl, 500 mM; with ethanol 1.6 Hydrolon, 1.2 μm KCl, 1 M; with ethanol2.1 Hydrolon, 1.2 μm KCl, 1.5 M; with ethanol 3.5 Hydrolon, 1.2 μmMgSO₄, 10 mM; with ethanol 1.9 Hydrolon, 1.2 μm MgSO₄, 100 mM; withethanol 4.6 Hydrolon, 1.2 μm MgSO₄, 500 M; with ethanol 2

EXAMPLE 19 Immobilization of Total RNA from an Aqueous Solution UsingVarious Buffer Conditions

[0248] Following the procedures of Example 1, plastic columns wereassembled using different hydrophobic membranes.

[0249] 100 μl of an aqueous solution containing total RNA were mixedwith 350 μl of a lysis buffer containing guanidinium isothiocyanate(concentration 2.5 M). The lysis buffer was mixed with variousconcentrations of sodium citrate, pH 7, or sodium oxalate, pH 7.2.Subsequently 250 μl of ethanol were added and mixed. This mixture wasthen transferred to a column and passed through the membrane and elutedaccording to the process described in Example 4. The results are listedin Table 12. Two tests were carried out to determine the average value.TABLE 12 RNA-yield from an aqueous solution with various bufferconcentrations in a binding solution Na-Citrate/Na-Oxalate, Yield ofTotal RNA Membrane Conc. in Lysis Buffer (μg) Hydrolon, 1.2 μmNa-Citrate, 10 mM 2.2 Hydrolon, 1.2 μm Na-Citrate, 100 mM 2.4 Hydrolon,1.2 μm Na-Citrate, 500 mM 3.55 Supor-450 PR Na-Citrate, 10 mM 1.1Supor-450 PR Na-Citrate, 100 mM 1.15 Supor-450 PR Na-Citrate, 500 mM 0.2Hydrolon, 1.2 μm Na-Oxalate, 1 mM 1.5 Hydrolon, 1.2 μm Na-Oxalate, 25 mM1.05 Hydrolon, 1.2 μm Na-Oxalate, 50 mM 0.9 Supor-450 PR Na-Oxalate, 1mM 1.9 Supor-450 PR Na-Oxalate, 25 mM 1.3 Supor-450 PR Na-Oxalate, 50 mM1.7

EXAMPLE 20 Immobilization of Total DNA from an Aqueous Solution UsingVarious Buffers

[0250] According to the procedures of Example 1, plastic columns wereassembled with hydrophobic membranes (for example Hydrolon 1.2 μm fromthe Pall Company).

[0251] 100 μl of an aqueous solution containing total DNA were mixedwith 350 μl of a lysis buffer containing guanidinium isothiocyanate (4 MGITC, 0.1 M MgSO₄). To this lysis buffer various buffer substances wereadded (concentration 25 mM) and adjusted to different pH-values.Subsequently, 250 μl of ethanol were added and mixed. The mixture wasthen introduced to the column and passed through the membrane, washedand eluted as in Example 4.

[0252] The results are set forth in Table 13. Triple tests are carriedout and average values determined. TABLE 13 DNA-yield from an aqueoussolution with various buffer substances in a binding solution BufferSubstance pH in the Lysis Buffer Yield of DNA (μg) Sodium Citrate pH 41.3 Sodium Citrate PH 5 0.6 Sodium Citrate pH 6 1.4 Sodium Citrate pH 70.5 Sodium Acetate pH 4 0.9 Sodium Acetate pH 5 1 Sodium Acetate pH 60.6 Sodium Acetate pH 7 0.5 Potassium Acetate pH 4 0.6 Potassium AcetatepH 5 0.9 Potassium Acetate pH 6 1.2 Potassium Acetate pH 7 1.4 AmmoniumAcetate pH 4 0.7 Ammonium Acetate pH 5 0.3 Ammonium Acetate pH 6 5.7Ammonium Acetate pH 7 1.5 Glycine pH 4 0.5 Glycine pH 5 1.1 Glycine pH 61.6 Glycine pH 7 1.1 Malonate pH 4 1.5 Malonate pH 5 0.3 Malonate pH 63.1 Malonate pH 7 1.6 Succinate pH 4 2.8 Succinate pH 5 2.3 Succinate pH6 2.5 Succinate pH 7 4.7

EXAMPLE 21 Immobilization of Total RNA from an Aqueous Solution UsingPhenol

[0253] According to the procedures of Example 1, plastic columns wereassembled with hydrophobic membranes (e.g., Hydrolon, 1.2 μm from thePall Company).

[0254] An aqueous solution containing RNA was mixed with 700 μl ofphenol and passed through the membranes using centrifugation. Themembranes were washed and the RNA eluted as in Example 4. Two tests werecarried out and an average value determined.

[0255] The amount of isolated RNA was subsequently determined byspectrophotometric measurement of the light absorption at a wavelengthof 260 nm and is on average 10.95 μg. The absorption ratio at 260 nm tothe one at 280 nm is 0.975.

EXAMPLE 22 Washing of Immobilized Total RNA under Different SaltConcentrations

[0256] Following the procedures of Example 1, plastic columns wereassembled with hydrophobic membranes.

[0257] 100 μl of an aqueous solution containing total RNA were mixedwith 350 μl of a lysis buffer containing guanidinium isothiocyanate(concentration 4 M). Subsequently, 250 μl of ethanol were added andmixed. This mixture was then transferred to the column and passedthrough the membrane and washed according to Example 4. The membraneswere then washed twice with a buffer containing various concentrationsof NaCl and 80% ethanol. The buffer was passed through the membrane bycentrifugation. The last washing step was carried out at 20000×g inorder to dry the membranes. The elution takes place according to theprocedure of Example 1. Two tests were carried out and an average valuedetermined. The results are listed in Table 14. TABLE 14 RNA-yield froman aqueous solution with NaCl in the washing buffer Yield of Total RNAMembrane NaCl in the Washing Buffer (μg) Hydrolon, 1.2 μm NaCl, 10 mM1.4 Hydrolon, 1.2 μm NaCl, 50 mM 3.15 Hydrolon, 1.2 μm NaCl, 100 mM 3Durapore, 0.65 μm NaCl, 10 mM 2.7 Durapore, 0.65 μm NaCl, 50 mM 2.85Durapore, 0.65 μm NaCl, 100 mM 2.7

EXAMPLE 23 Elution of Immobilized Total RNA under Different Salt andBuffer Conditions

[0258] According to the procedures of Example 1, plastic columns wereassembled with hydrophobic membranes.

[0259] 100 μl of an aqueous solution containing total RNA were mixedwith 350 μl of a lysis buffer containing guanidinium isothiocyanate(concentration 4 M). Subsequently, 250 μl of ethanol were added andmixed. This mixture was then introduced to the column and passed throughthe membrane and washed according to the procedures of Example 4.

[0260] For elution, 70 μl of a NaCl-containing solution, a Tris/HClbuffer (pH 7) or a sodium oxalate solution (pH 7.2) were pipetted ontothe membrane, in order to elute the purified RNA from the membrane.After 1 to 2 minutes of incubation at a temperature of 10° C.-30° C.,the eluate was pipetted from above from the membrane. The elution stepwas repeated once in order to achieve complete elution. Two tests werecarried out and an average value determined. The results are summarizedin Table 15. TABLE 15 RNA-yield from an aqueous solution with NaCl,Tris/HCl or sodium oxalate in the elution buffer NaCl or Tris Membranein the Elution Buffer Yield of Total RNA (μg) Hydrolon, 1.2 μm NaCl, 1mM 1.35 Hydrolon, 1.2 μm NaCl, 50 mM 1.2 Hydrolon, 1.2 μm NaCl, 250 mM0.45 Durapore, 0.65 μm NaCl, 1 mM 0.9 Durapore, 0.65 μm NaCl, 50 mM 0.35Durapore, 0.65 μm NaCl, 500 mM 0.15 Hydrolon, 1.2 μm Tris/HCl, 1 mM 0.35Hydrolon, 1.2 μm Tris/HCl, 10 mM 0.75 Durapore, 0.65 μm Tris/HCl, 1 mM1.5 Durapore, 0.65 μm Tris/HCl, 50 mM 1 Durapore, 0.65 μm Tris/HCl, 250mM 0.1 Hydrolon, 1.2 μm Na-Oxalate, 1 mM 0.45 Hydrolon, 1.2 μmNa-Oxalate, 10 mM 0.65 Hydrolon, 1.2 μm Na-Oxalate, 50 mM 0.3 Durapore,0.65 μm Na-Oxalate, 1 mM 2 Durapore, 0.65 μm Na-Oxalate, 10 mM 0.155Durapore, 0.65 μm Na-Oxalate, 50 mM 0.15

EXAMPLE 24 Elution of the Immobilized RNA at Different Temperatures

[0261] Following the procedure of Example 1, plastic columns wereassembled using a hydrophobic membrane (e.g., Hydrolon, 3 μm from thePall Company).

[0262] For isolation, 5×10⁵ HeLa-cells were used. The followingisolation steps were carried out as described in Example 3.

[0263] For elution, 70 μl of RNase-free water of a different temperaturewere pipetted onto the membrane in order to elute the purified RNA fromthe membrane. After an incubation of 1-2 minutes at the correspondingelution temperature, the eluate was pipetted off the membrane fromabove. The elution step was repeated once in order to achieve completeelution. Triple tests were carried out and an average value determined.The results are summarized in Table 16. TABLE 16 RNA-yield at differentelution temperatures Membrane Elution Temperature Yield of Total RNA(μg) Hydrolon, 3 μm Ice cold 2.2 Hydrolon, 3 μm 40° C. 3.2 Hydrolon, 3μm 50° C. 3.9 Hydrolon, 3 μm 60° C. 3.7 Hydrolon, 3 μm 70° C. 3.7Hydrolon, 3 μm 80° C. 2.9

EXAMPLE 25 Elution of Immobilized RNA by Way of Centrifugation

[0264] Following the procedures of Example 1, plastic columns wereassembled with a hydrophobic membrane (e.g., Hydrolon 1.2 μm from thePall Company).

[0265] 100 μl of an aqueous solution containing total RNA were mixedwith 350 μl of a commercially available lysis buffer containingguanidinium isothiocyanate (e.g., RLT buffer from QIAGEN). 250 μl ofethanol were then added and mixed by pipetting. This mixture was thentransfered onto the column and passed through the membrane usingcentrifugation (10000×g; 1 minute). The membranes were subsequentlywashed twice with a buffer (e.g., RPE buffer from QIAGEN). Each time thebuffer was passed through the membranes by way of centrifugation. Thelast washing step was carried out at 20000×g in order to dry themembrane.

[0266] For elution, 70 μl of RNase-free water were pipetted onto themembrane in order to elute the RNA from the membrane. After anincubation of 1 minute at a temperature of 10° C.-30° C., the eluate waspassed through the membrane by centrifugation (10000×g, 1 minute). Inorder to achieve complete elution, the elution step was repeated onceand the eluates joined together. Five parallel tests were carried outand the average value calculated.

[0267] The amount of isolated total RNA was subsequently determined byspectrophotometric measurement of the light absorption at a wavelengthof 260 nm and was on average 6.4 μg. The absorption ratio at 260 nm to280 nm was 1.94.

EXAMPLE 26 Use of Total RNA in a ‘Real Time’ Quantitative RT-PCR Using5′ Nuclease PCR Assay to Amplify and Detect β-actin mRNA

[0268] Following the procedures of Example 3, plastic columns wereassembled using a commercially available membrane (Hydrolon from Pall,with a pore size of 3 μm).

[0269] To isolate RNA, 1×10⁵ HeLa cells were used, and the purificationof total RNA was carried out as described in Example 1. The elution wascarried out with 2×70 μl of H₂O as described in Example 1. For thecomplete removal of remaining amounts of DNA, the sample was treatedwith a DNase prior to analysis.

[0270] A “one-device ‘Real Time’ quantitative RT-PCR” was carried outwith the use of the commercially available reaction system ofPerkin-Elmer (TaqMan™ PCR Reagent Kit) by using a M-MLV reversetranscriptase. By using a specific primer and a specific TaqMan probefor β-actin (TaqMan™ β-actin Detection Kit, made by Perkin Elmer) theβ-actin mRNA molecules in the total RNA sample were first converted intoβ-actin cDNA and subsequently the total reaction was amplified anddetected immediately, without interruption, in the same reaction device.The reaction specimens were produced according to the manufacturer'sinstructions. Three different amounts of isolated total RNA are used (1,2, 4 μl of eluate) and triple determination tests were carried out. As acontrol, three specimens without RNA were also tested.

[0271] The cDNA synthesis was carried out at 37° C. for one hour,immediately followed by a PCR which comprised 40 cycles. The reactionsand the analyses were carried out on an ABI PRIS™ 7700 Sequence Detectormanufactured by Perkin Elmer Applied Biosystems. Every amplicongenerated during a PCR-cycle produces a light emitting molecule, whichis generated by splitting from the TaqMan-probe. The total light signalthat is generated is directly proportional to the amplicon quantity thatis being generated and hence to the original amount of transcriptavailable in the total RNA sample. The emitted light is measured by theinstrument and evaluated by a computer program. The PCR cycle, duringwhich the light signal must first be detected over the background noise,will be designated as the “Threshold Cycle” (ct). This value is ameasure for the amount of specifically amplified RNA available in thesample.

[0272] For the 1 μl RNA eluate, isolated with the process describedhere, an average ct-value of 17.1 was calculated; for 2 μl in total RNAthe ct-value was 16.4 and for 4 μl of total RNA the ct-value was 15.3.This resulted in a linear dependency between the total RNA and thect-value, indicating that the total RNA was free of substances thatmight inhibit the amplification reaction. The control specimenscontaining no RNA did not produce any signals.

EXAMPLE 27 Use of Total RNA in an RT-PCR for Amplification and Detectionof β-actin mRNA

[0273] According to Example 1, plastic columns were assembled withcommercially available membranes (Pall, Hydrolon with a pore size of 1.2or 3 μm; Sartorius, Sartolon with a pore size of 0.45 μm).

[0274] For isolation of RNA, two different starting materials were used:(1) total RNA from liver (mouse) in an aqueous solution; purification,elution carried out as described in Example 4; and (2) 5×10⁵ HeLa-cells,the purification of total RNA and the elution are carried out asdescribed in Example 3.

[0275] For each test, 20 ng of isolated total RNA were used. As acontrol, RNA which was purified by way of RNeasy-Kits (QIAGEN) and asample without RNA were used.

[0276] A RT-PCR was performed with these samples under standardconditions. For amplification two different primer pairs were used forthe β-actin-mRNA. A 150 bp-sized fragment serves as proof ofsensitivity, a 1.7 kbp-sized fragment assesses the integrity of the RNA.From the RT-reaction, 1 μl was removed and introduced to the subsequentPCR. 25 cycles were performed for the small fragment and 27 cycles forthe large fragment. The annealing temperature was 55° C. The amplifiedsamples were subsequently placed on a non-denaturing gel and analyzed(FIG. 8).

[0277] For the 20 ng quantity used of total RNA isolated in the processdescribed above, the corresponding DNA-fragments can be demonstrated inthe RT-PCR. When using total RNA from mouse liver, no transcript can bedemonstrated, as the conditions used here are adjusted to human β-actinmRNA. The control specimens which contain no RNA do not produce anysignals.

[0278]FIG. 8 shows ethidium bromide stained agarose gels of anelectrophoretic separation of RT-PCR reaction products.

[0279]FIG. 8A: Lanes 1 to 8: RT-PCR of the 150 bp fragment:

[0280] Lanes 1 & 2: RNA from mouse liver in an aqueous solution purifiedwith the Hydrolon 1.2 μm membrane;

[0281] Lanes 3 & 4: RNA from HeLa-cells purified with the Sartolonmembrane;

[0282] Lanes 5 & 6: RNA from HeLa-cells purified with the Hydrolon 3 μmmembrane;

[0283] Lane 7: RNA purified using the RNeasy-Mini-Kit;

[0284] Lane 8: Control without RNA.

[0285]FIG. 8B: Lanes 1 to 8: RT-PCR of the 1.7 kbp fragment:

[0286] Lanes 1 & 2: RNA from mouse liver in an aqueous solution purifiedwith the Hydrolon 1.2 μm membrane;

[0287] Lanes 3 & 4: RNA from HeLa-cells purified with the Sartolonmembrane;

[0288] Lanes 5 & 6: RNA from HeLa-cells purified with the Hydrolon 3 μmmembrane;

[0289] Lane 7: RNA purified using the RNeasy-Mini-Kit;

[0290] Lane 8: Control without RNA.

EXAMPLE 28 Use of Total RNA in a NASBA-reaction (Nucleic Acid SequenceBased Amplification) for the Amplification and Detection of β-actin mRNA

[0291] Following the procedures described in Example 1, plastic columnswere assembled with commercially available membranes (Pall, Hydrolonwith a pore size of 1.2 or 3 μm; Sartorius, Sartolon with a pore size of0.45 μm). For isolation of RNA, two different starting materials wereused: (1) total RNA from liver (mouse) in an aqueous solution;purification, elution carried out as described in Example 4; and (2)5×10⁵ HeLa-cells, the purification of total RNA and the elution arecarried out as described in Example 3.

[0292] A NASBA-reaction is performed under standard conditions (Fahy, E.et al., 1991, PCR Methods Amplic., 1:25-33). For amplification, β-actinspecific primers were used.

[0293] For each test 20 ng of isolated total RNA are used. As a control,RNA which was purified by way of RNeasy-Kits (QIAGEN) and a samplewithout RNA, were used. First they were incubated for 5 minutes at 65°C. and for 5 minutes at 41° C. Following this step, an enzyme mixtureconsisting of RNaseH, T7-polymerase and AMVV-RT was added and incubatedfor 90 minutes at 41° C. The amplified samples were subsequently placedon a non-denaturing gel and analyzed. For the 20 ng of total RNAisolated in the process described above, a specific transcript can bedemonstrated (FIG. 9).

[0294]FIG. 9 shows an ethidium-bromide stained agarose gel of anelectrophoretic separation of the NASBA-reactions.

[0295] Lanes 1 to 8: NASBA-Reactions:

[0296] Lanes 1 & 2: RNA from mouse liver purified from an aqueoussolution with the 1.2 μm Hydrolon membrane;

[0297] Lane 3 & 4: RNA from HeLa-cells purified with the Sartolonmembrane;

[0298] Lane 5 & 6: RNA from HeLa-cells purified with the 3 μm Hydrolonmembrane;

[0299] Lane 7: RNA purified using the RNeasy-Mini-Kit;

[0300] Lane 8: Control without RNA.

EXAMPLE 29 NASBA-reaction for Amplification and Detection of β-actinmRNA on Hydrophobic Membranes

[0301] According to the procedures of Example 1, plastic columns wereassembled with commercially available membranes (Pall, Hydrolon with apore size of 3 μm; Supor-450 PR with a pore size of 0.45 μm; Millipore,Fluoropore with a pore size of 3 μm).

[0302] For the isolation of RNA, different quantities of HeLa cells wereused, the purification of total RNA was carried out as described inExample 3. The elution was performed by adding 20 μl NASBA-reactionbuffer. The NASBA-reaction is subsequently performed on the membrane.

[0303] A NASBA-reaction is performed under standard conditions (Fahy, E.et al., 1991, PCR Methods Amplic., 1:25-33). For amplification, β-actinspecific primers were used.

[0304] The reaction device was first incubated for 5 minutes at 41° C.in a water bath. Following this step, an enzyme mixture consisting ofRNaseH, T7-Polymerase and AMVV-RT was added and incubated for 90 minutesat 41° C. The amplified samples subsequently were placed on anon-denaturing gel and analyzed. For the quantity of RNA used andisolated from 5×10⁵ to 3×10⁴ HeLa cells, a specific transcript can beobserved for the total RNA isolated by the process described here.

[0305]FIG. 10 shows an ethidium-bromide stained agarose gel of anelectrophoretic separation of the NASBA-reactions.

[0306]FIG. 10A: Lanes 1 to 4: RNA from HeLa-cells purified with the 3 μmHydrolon membrane:

[0307] Lane 1: 2.5×10⁵ cells;

[0308] Lane 2: 1.25×10⁵ cells;

[0309] Lane 3: 6×10⁴ cells;

[0310] Lane 4: 3×10⁴ cells.

[0311]FIG. 10B: Lanes 1 to 3: RNA purified from HeLa-cells:

[0312] Lane 1: RNA from 2.5×10⁵ HeLa-cells purified with the 3 μmHydrolon membrane;

[0313] Lane 2: RNA from 5×10⁵ HeLa-cells purified with the Supor-450 PRmembrane;

[0314] Lane 3: RNA from 5×10⁵ HeLa-cells purified with the 3 μmFluoropore membrane;

EXAMPLE 30 Restriction of Plasmid DNA with the Ava I enzyme on aHydrophobic Membrane

[0315] According to the procedures of Example 1, plastic columns wereassembled with hydrophobic membranes (e.g., Supor-200 PR from Pall).

[0316] 100 μl of a plasmid-containing aqueous solution (PCMVβ byClontech) were mixed with 350 μl of a lysis buffer containingguanidinium isothiocyanate (4 M GITC, 0.1 M MgSO₄, 25 mM sodium acetate,pH 4). Subsequently, 250 μl of isopropanol were added and mixed bypipetting. This mixture was then introduced to the column and passedthrough the membrane, washed and dried according to Example 4.

[0317] 100 μl of a 1×buffer for the restriction enzyme Ava I were placedon the membrane and either: (1) removed, transferred to a new reactiondevice and subsequently treated with the restriction enzyme (i.e., Ava Iby Promega); or (2) a restriction enzyme (i.e., Ava I by Promega) wasadded directly to the eluate in the column. The reaction mixtures wereincubated for 1 hour at 37° C. and subsequently placed on anon-denaturing gel and analyzed (see FIG. 11).

[0318]FIG. 11 shows an ethidium-bromide stained agarose gel of anelectrophoretic separation of pCMVβ-plasmid after restriction with Ava I

[0319] Lane 1: uncut plasmid;

[0320] Lanes 2 & 3: elution with the reaction buffer for Ava I,restriction reaction in a separate device;

[0321] Lane 4 & 5: restriction with Ava I on the membrane.

EXAMPLE 31 Pressure Filtration for Isopropanol Precipitation of DNA

[0322] The isolation of plasmid DNA was performed according to standardprotocols including the elution step via anion exchange chromatography.The DNA was eluted from the column in a high saline buffer.

[0323] Subsequently, 0.7 volume of isopropanol was added to this DNAsolution, the sample was mixed and incubated for 1-5 minutes at roomtemperature. A 0.45 μm cellulose acetate filter with a 5 cm² surface ina filtration cartridge (standard installation for sterile filtration,e.g., Minisart by Sartorius) was used as a filtration installation. Thisfilter was connected to a syringe from which the plunger has beenremoved first. The syringe was then filled with the DNA/isopropanolmixture and pressed through the filter with the syringe plunger. A highpercentage of the DNA in this precipitate stays on the filter (i.e.,cannot pass the pores).

[0324] The plunger was again removed from the syringe, was insertedagain, and air was pressed through the filter. This step was repeatedonce or twice and serves to dry the membrane.

[0325] Subsequently, elution was performed with a corresponding volumeof a low saline buffer, whereby the buffer fills the syringe and waspressed through the filter with the plunger. To increase the yield, thisfirst eluate was again put into the syringe and pressed through thefilter with the plunger. In this test configuration, the yields obtainedtypically range from 80 to 90% (see Example 34).

EXAMPLE 32 Vacuum Filtration for the Isopropanol Precipitation of DNA

[0326] As with pressure filtration, first plasmid DNA was isolated andmixed with 0.7 volume isopropanol. An apparatus designed for vacuumfiltration was used as a filtration installation, in which a 0.45 μmcellulose acetate filter with a surface of 5 cm² was placed. 0.45 μmcellulose nitrate filters or several layers of 0.65 μm cellulose acetateor cellulose nitrate filters may be used. The isopropanol-DNA mixturewas incubated for 1-5 minutes and placed on the filter assembly. Bycreating a vacuum, the solution was suctioned through the filter. TheDNA-precipitates on the filter were mixed with a corresponding volume of70% ethanol and washed by creating a vacuum. The elution of the DNA fromthe filter takes place by adding a low salt buffer, a short incubationand renewed creation of a vacuum. The yield can either be obtained byrepeated elution from the filter with a second volume of low salinebuffer or by elution with the eluate from the first elution step. Herealso, typical yields range from 80%-90% of the DNA

EXAMPLE 33

[0327] The method used is the vacuum filtration method described inExample 32. The filter device used is the vacuum filter apparatus,Sartorius 16315. pCMVβ was used as the plasmid DNA, which was isolatedfrom DH5α cells.

[0328] Procedure: In each test, 15 ml of QF-buffer (high saline buffer)are mixed with 500 μg of plasmid. 10.5 ml of isopropanol are added andthis is mixed again. Then the mixture is left to incubate for 5 minutes.The plasmid DNA thus precipitated is deposited on the membrane in thefilter assembly. Next a vacuum is created and the filtration takesplace. The membranes are washed with 5 ml of 70% ethanol (by creatinganother vacuum), then 1 ml TE-buffer is pipetted onto the membranes,left to incubate for 5 minutes, and the DNA is eluted by creating avacuum. Subsequently a post-elution is performed with 1 ml TE-buffer.Total DNA amounts are measured in the flow-through, in the washing stageand in the combined eluate (OD260). The following results were obtained:Membrane Test Number Flow-through Washing Stage Eluate Flow Speed PVDF0.2 μm 1 0 μg DNA 0 μg DNA 131 μg Very slow DNA Cellulose Nitrate 2 0 μgDNA 0 μg DNA 418 μg Fast 0.65 μm DNA Cellulose Acetate 3 0 μg DNA 0 μgDNA 469 μg Fast 0.65 μm DNA

[0329] Calculated on the basis of 500 μg of DNA starting quantity, thefollowing yields are obtained with this method: PVDF 0.2 μm 26%Cellulose Acetate 0.65 μm 94% Cellulose Nitrate 0.65 μm 84%

EXAMPLE 34

[0330] The pressure filtration method indicated in Example 31 was used.The filter assembly used was a commercially available 0.45 μm celluloseacetate filter (Minisart, Sartorius). pCMV β is used as plasmid DNA,which was isolated from DH5α cells.

[0331] Procedure: For each test, 15 ml of QF-buffer (high salt buffer)are added to and mixed with 100, 200, 300, etc., up to 900 μg ofplasmid. 10.5 ml isopropanol are added and again mixed. Subsequently,there is a 5-minute incubation period. The plasmid DNA thus precipitatedis transferred to a syringe, to which the filter had been previouslyfitted. Pressure filtration takes place with the aid of the syringe. Thefilter is then washed with 2 ml of 70% ethanol and, as described, driedtwice. The elution is performed with 2 ml of TE-buffer. A second elutionis performed with the eluate. The total amount of DNA is measured in thecombined eluate (OD260).

[0332] Following the above procedure, the following results wereobtained: DNA-quantities used DNA-quantities eluted % Yield 100 μg 100μg 100% 200 μg 176 μg 88% 300 μg 257 μg 86% 400 μg 361 μg 90% 500 μg 466μg 93% 600 μg 579 μg 97% 700 μg 671 μg 96% 800 μg 705 μg 88% 900 μg 866μg 96%

EXAMPLE 35

[0333] The vacuum filtration method indicated in Example 32 was used.The filter assembly used was a commercially obtained 0.45 μm celluloseacetate filter (Minisart, Sartorius), that had been attached to afiltration chamber (QIAvac). As buffer reservoir, a syringe was attachedto the other end of the filter. pCMVβ was used as plasmid DNA, which wasisolated from DH5α cells.

[0334] Procedure: 15 ml of QF-buffer (high saline buffer) are added toand mixed with 500 μg of plasmid. 10.5 ml isopropanol are added andagain mixed. Subsequently, there is a 5-minute incubation period. Theplasmid DNA thus precipitated is then transferred to the filter assemblysyringe. Now a vacuum is created and filtration takes place. The filteris not washed with 70% ethanol. Rather, elution with 2 ml of EB buffer(QIAGEN) follows immediately. Post-elution is performed with the eluate.The total DNA quantity in the combined eluate is measured (OD260). Thefollowing result was obtained: Test Number Eluted DNA % Yield 1 434 μg87% 2 437 μg 87%

[0335] Although a number of embodiments have been described above, itwill be understood by those skilled in the art that modifications andvariations of the described devices and methods may be made withoutdeparting from concept of the invention as defined in the appendedclaims. The articles and other publications cited herein areincorporated by reference.

1. A process for the isolation of nucleic acids from a sample includingthe following steps: (a) applying at least one nucleic acid sample to amembrane; (b) immobilizing the nucleic acids on the membrane; (c)releasing the immobilized nucleic acids from the membrane; and (d)removing the released nucleic acids through the membrane, whereby themembrane is comprised of one or more materials selected from the groupconsisting of nylon, polysulfone, polyethersulfone, polycarbonate,polyacrylate, acrylic copolymer, polyurethane, polyamide,polyvinylchloride, polyfluorocarbonate, poly-tetrafluoro-ethylene,polyvinylidene fluoride,polyethylene-tetrafluoro-ethylene-copolymerisate, polybenzimidazole,polyethylene-chlorotrifluoro-ethylene-copolymerisate, polyimide,polyphenylene sulfide, cellulose, cellulose-mix ester, cellulosenitrate, cellulose acetate, polyacrylnitrile, polyacrylnitril-copolymer,nitrocellulose, polypropylene and polyester.
 2. The process according toclaim 1, characterized in that the nucleic acid sample is applied on topof the membrane and the nucleic acids are removed from below themembrane.
 3. The process according to claims 1 or 2, characterized inthat the membrane is placed in a container with an inlet and outlet andthe membrane fills the entire cross-section of the container, separatingsaid inlet and outlet.
 4. The process according to claim 1,characterized in that the membrane is coated.
 5. The process accordingto claim 4, characterized in that the membrane has been made hydrophobicby the coating.
 6. The process according to claim 4, characterized inthat the membrane has been made hydrophilic by the coating.
 7. Theprocess according to claim 1, characterized in that the membrane is lessthan 1 mm thick.
 8. The process according to claim 7, characterized inthat the membrane is less than 0.5 mm.
 9. A process for the isolation ofnucleic acids from a sample comprising the following steps: (a) applyingat least one nucleic acid sample to a surface; (b) immobilizing thenucleic acids on the surface; (c) releasing the immobilized nucleicacids from the surface with an elution agent, characterized in that therelease takes place at a temperature T, whereby 10° C.≧T≧_(S,EM,) andT_(S,EM) equals the freezing point of the elution agent.
 10. The processaccording to claim 9, characterized in that the release takes place attemperature T, in which 10° C.≧T≧5° C.
 11. The process according toclaims 9, characterized in that the release takes place at temperatureT, in which 10° C.≧T≧0° C.
 12. The process according to claims 9,characterized in that the release takes place at temperature T, in which10° C.≧T≧−5° C.
 13. The process according to claim 9, characterized inthat the release takes place at temperature T, in which 5°C.≧T≧T_(S,EM).
 14. A process for the isolation of nucleic acids from asample comprising the following steps: (a) adjusting a nucleic acidsample to binding conditions that permit immobilization of the nucleicacids contained in the sample on a surface; (b) applying the nucleicacids sample to the surface; and (c) immobilizing the nucleic acids onthe surface, characterized in that, before and/or after adjusting thebinding conditions there is a pre-treatment of the sample.
 15. Theprocess according to claim 14, characterized in that the pre-treatmenttakes place by salting out.
 16. The process according to claim 14,characterized in that the pre-treatment takes place by way offiltration, centrifugation, enzymatic treatment, temperature effect,precipitation, extraction, homogenization, mechanical reduction and/orbinding of contaminants to surfaces.
 17. The process according to claim14, characterized in that the binding conditions permit immobilizationof RNA.
 18. The process according to claim 14, characterized in that thebinding conditions permit immobilization of DNA.
 19. The processaccording to claim 14, characterized in that the following additionalsteps are included: releasing of immobilized nucleic acids from thesurface, and removing the released nucleic acids from the surface. 20.The process according to any one of claims 1, 9 or 14, characterized inthat after the release step at least one additional step takes place:performing at least one chemical reaction with the nucleic acids.
 21. Aprocess for performing a nucleic acid amplification reaction comprisingfollowing steps: (a) applying at least one nucleic acid sample to asurface; (b) immobilizing the nucleic acids on the surface; and (c)performing an amplification reaction with the nucleic acids.
 22. Theprocess according to claim 21, characterized in that the amplificationreaction is not isothermal.
 23. The process according to claim 21,characterized in that the amplification reaction is isothermal.
 24. Theprocess according to claim 21, wherein the nucleic acid amplificationreaction is a Strand Displacement Amplification (SDA) reaction, a PCR,or an RT-PCR.
 25. The process according to claim 21, characterized inthat, prior to performing the amplification reaction, the nucleic acidsare released with a suitable reaction buffer from the surface and theeluate is located on or in the membrane.
 26. The process according toclaim 25, characterized in that there is an additional step of removingthe released amplification reaction products from the surface.
 27. Theprocess according to claim 21, characterized in that the nucleic acidamplification reaction takes place in a reaction buffer that does notresult in release of the nucleic acids from the surface.
 28. The processaccording to claim 27, which includes the additional steps of: (d)releasing the amplification reaction products from the surface; and (e)removing the released amplification reaction products from the surface.29. A process for performing chemical reactions on nucleic acidsincluding the following steps: (a) applying at least one nucleicacid-containing sample to a surface; (b) immobilizing the nucleic acidson the surface; (c) releasing the immobilized nucleic acids from thesurface; (d) performing at least one chemical reaction with the nucleicacids; and (e) removing the nucleic acids from the surface withoutadditional immobilization.
 30. A process for analysis of nucleic acidsin an isolation device including the following steps: (a) providing anisolation device with a membrane located therein; (b) applying at leastone nucleic acid-containing sample to the isolation device; (c)immobilizing the nucleic acids on the membrane; (d) passing the liquidcomponents of the sample through the membrane; and (e) analyzing atleast one property of the nucleic acid on the membrane located in theisolation device.
 31. The process according to claim 30, characterizedin that after passing the liquid components through the membrane, atleast one chemical reaction is performed with the nucleic acids.
 32. Theprocess according to claim 31, characterized in that the chemicalreaction is a radioactive labeling of the nucleic acid.
 33. The processaccording to claim 30, characterized in that the analyzed property isthe binding capacity of the nucleic acids for molecules.
 34. The processaccording to claim 33, characterized in that the molecules areantibodies.
 35. The process according to claim 33, characterized in thatthe molecules are nucleic acid binding proteins.
 36. The processaccording to claim 33, characterized in that the molecules are dyemolecules.
 37. The process according to any one of claims 9, 14, 21, 29or 30, characterized in that the sample is introduced onto the top ofthe membrane or surface.
 38. A process according to any one of claims 1,9, 19, 28, 29, or 30, characterized in that the immobilized nucleicacids are subjected to a washing step which takes place with at leastone washing buffer after the immobilization and before any releasesteps.
 39. The process according to claim 38, characterized in that thewashing step consists of the following steps for each washing buffer:applying a predetermined quantity of washing buffer on the surface; andpassing the washing buffer through the surface.
 40. The processaccording to any one of claims 1, 9, 19, 28, or 29, characterized inthat an aqueous salt or buffer solution is used to release the nucleicacids.
 41. The process according to any one of claims 1, 9, 19, 28, or29, characterized in that water is used to release the nucleic acids.42. The process according to one of claims 1, 9, 14, 21, 29, or 30,characterized in that the introduction and immobilization of the nucleicacids includes the following steps: (a)(1) mixing at least one nucleicacid-containing sample with an immobilization buffer; (a)(2) applyingsaid at least one nucleic acid-containing sample with the immobilizationbuffer to the surface or membrane; and (a)(3) passing the liquidcomponents through the surface in essentially the same direction theywere added.
 43. The process according to any one of claims 1, 9, 19, 28,29 or 30, characterized in that at least one of the steps is carried outby an automatic device, in a fully automatic manner.
 44. The processaccording to claim 43, characterized in that all steps of the processare performed by an automatic apparatus in a controlled sequence. 45.The process according to claim 43, characterized in that a majority ofnucleic acid isolations or reactions take place simultaneously.
 46. Theprocess according to any one of claims 1, 9, 19, 28, 29, or 30,characterized in that aqueous salt solutions of metal and/or ammoniumcations with mineral acids are used to immobilize the nucleic acids. 47.The process according to claim 46, wherein the aqueous salt solutionsare of alkaline halides, alkaline-earth halides, alkaline sulfates,alkaline-earth sulfates, alkaline phosphates, alkaline-earth phosphates,or mixtures thereof.
 48. The process according to claim 47,characterized in that sodium halides, lithium halides and/or potassiumhalides and/or magnesium sulfate are used to immobilize the nucleicacids.
 49. The process according to any one of claims 1, 9, 19, 28, 29,or 30, characterized in that aqueous solutions of salts of mono orpolybasic or polyfunctional organic acids with alkaline oralkaline-earth metals are used to immobilize the nucleic acids.
 50. Theprocess according to claim 49, characterized in that aqueous solutionsof sodium, potassium or magnesium salts with organic dicarboxylic acidsare used to immobilize the nucleic acids.
 51. The process according toclaim 50, characterized in that the organic dicarboxylic acid is oxalicacid, malonic acid and/or succinic acid.
 52. The process according toclaim 49, characterized in that aqueous solutions of sodium or potassiumsalts with a hydroxy or polyhydroxycarboxylic acid are used toimmobilize the nucleic acids.
 53. The process according to claim 52,characterized in that the polyhydroxycarboxylic acid is citric acid. 54.The process according to any one of claims 1, 9, 19, 28, 29, or 30,characterized in that hydroxy-functional compounds of aliphatic oracyclic saturated or unsaturated hydrocarbons are used for theimmobilization of the nucleic acids.
 55. The process according to claim54, wherein said hydroxy-functional compounds are selected from theC₁-C₅ alkanols.
 56. The process according to claim 55, wherein saidalkanols are selected from methanol, ethanol, n-propanol, tert.-butanol,pentanols, and mixtures thereof.
 57. The process according to claim 54,wherein said hydroxy-functional compound is an aldite.
 58. The processaccording to any one of claims 1, 9, 19, 28, 29, or 30, characterized inthat a phenol or polyphenol is used for the immobilization of thenucleic acids.
 59. The process according to any one of claims 1, 9, 19,28, 29, or 30, wherein at least one chaotropic agent is used for theimmobilization of the nucleic acids.
 60. The process according to claim59, characterized in that the chaotropic agent is a salt selected fromthe group of trichloracetates, thiocyanates, perchlorates, iodides,guanidinium hydrochloride, guanidinium isothiocyanate, and urea.
 61. Theprocess according to claim 59, characterized in that 0.01 molar to 10molar aqueous solutions of at least one chaotropic agent by itself, orin combination with other salts, is used to immobilize the nucleicacids.
 62. The process according to claim 61, characterized in that 0.1molar to 7 molar aqueous solutions of at least one chaotropic agent byitself, or in combination with other salts, is used to immobilize thenucleic acids.
 63. The process according to claim 62, characterized inthat 0.2 molar to 5 molar aqueous solutions of at least one chaotropicagent by itself, or in combination with other salts, is used toimmobilize the nucleic acids.
 64. The process according to any one ofthe claims 61 to 63, wherein the chaotropic agent is selected from anaqueous solution of one or more of sodium perchlorate, guanidiniumhydrochloride, guanidinium isothiocyanate, sodium iodide and potassiumiodide.
 65. The process according to claim 38, wherein washing steps arecarried out using salt or buffer solutions selected from aqueous saltsolutions of metal and/or ammonium cations with mineral acids, includingalkaline halides, alkaline-earth halides, alkaline sulfates,alkaline-earth sulfates, alkaline phosphates, alkaline-earth phosphates,or mixtures thereof; aqueous solutions of salts of mono or polybasic orpolyfunctional organic acids with alkaline or alkaline-earth metals,including sodium, potassium or magnesium salts of organic dicarboxylicacids including oxalic acid, malonic acid and succinic acid; aqueoussolutions of sodium or potassium salts of a hydroxy orpolyhydroxycarboxylic acid including citric acid; hydroxy-functionalcompounds of aliphatic or acyclic saturated or unsaturated hydrocarbonsincluding C₁-C₅ alkanols and aldites; phenols ir polyphenols; one ormore chaotropic agents including salts selected from the group oftrichloracetates, thiocyanates, perchlorates, iodides, guanidiniumhydrochloride, guanidinium isothiocyanate, and urea.
 66. The processaccording to any one of claims 9, 14, 19, 21, 28, or 29, characterizedin that the surface is a membrane.
 67. The process according to claim66, characterized in that the membrane is a hydrophobic membrane. 68.The process according to claim 67, characterized in that the hydrophobicmembrane consists of a polymer with polar groups.
 69. The processaccording to claim 67 or 68, characterized in that the membrane is ahydrophilic membrane with a hydrophobic surface.
 70. The processaccording to claim 67 or 68, characterized in that the membrane is madeof nylon, a polysulfone, polyethersulfone, polycarbonate, polypropylene,polyacrylate, acrylic copolymer, polyurethane, polyamide,polyvinylchloride, polyfluorocarbonate, poly-tetrafluoro-ethylene,polyvinylidene fluoride,polyethylene-tetrafluoro-ethylene-copolymerisate, apolyethylene-chlorotrifluoro-ethylene-copolymerisate, cellulose acetate,nitrocellulose, polybenzimidazole, polyimide, polyacrylnitrile,polyacrylnitrile-copolymer, cellulose-mix ester, cellulose nitrate, orpolyphenylene sulfide.
 71. The process according to claim 70,characterized in that the membrane consists of hydrophobic nylon. 72.The process according to claim 71, characterized in that the membrane iscoated with a hydrophobizing coating agent selected from the group ofparaffins, waxes, metal soaps, optionally containing additives selectedfrom the group of aluminum or zirconium salts, quaternary organiccompounds, ureic derivates, lipid modified resins, silicones, zincorganic compounds and glutaric dialdehyde.
 73. The process according toclaim 1, wherein the membrane is a hydrophilic membrane or a membranemade hydrophilic by pre-treatment.
 74. The process according to claim 73characterized in that the membrane consists of hydrophilisized nylon,polyethersulfone, polycarbonate, polyacrylate, acrylic copolymer,polyurethane, polyamide, polyvinylchloride, polyfluorocarbonate,poly-tetrafluoro-ethylene, polyvinylidene fluoride,polyethylene-tetrafluoro-ethylene-copolymerisate, apolyethylene-chlorotrifluoro-ethylene-copolymerisate, cellulose acetate,polypropylene, nitrocellulose, polybenzimidazole, polyimide,polyacrylnitrile, polyacrylnitrile-copolymer, cellulose-mix ester,polyester, polysulfone, cellulose nitrate, or polyphenylene sulfide. 75.The process according to any one of claims 9, 14, 19, 21, 28, or 29,characterized in that the membrane has a pore diameter of 0.001 to 50micrometer.
 76. The process according to any one of claims 9, 14, 19,21, 28, or 29, characterized in that the surface is a hydrophobicfleece.
 77. A process for isolating nucleic acids including thefollowing steps: (a) providing an isolation device with at least onemembrane located therein; (b) applying a nucleic acid-containing sampleto the isolation device; (c) precipitating the nucleic acids containedin the sample with an alcohol, so that the nucleic acids are bound tothe at least one membrane, characterized in that the pore size of saidat least one membrane is equal or larger than 0.2 micrometer.
 78. Theprocess according to claim 77, characterized in that the alcohol isadded to the nucleic acid-containing sample prior to adding the sampleto the isolation device.
 79. The process according to claim 77,characterized in that the alcohol is added to the nucleicacid-containing sample after adding the sample to the isolation device.80. The process according to claim 77, characterized in that the surfaceof the membrane is selected so that all the nucleic acids contained inthe solution can be bound to the membrane.
 81. The process according toclaim 77, wherein said membrane has a pore size equal to or greater than0.2 micrometer.
 82. The process according to claim 81, wherein saidnucleic acids precipitated are DNA and/or RNA.
 83. The process accordingto claim 77, wherein the alcohol used is a C₁-C₅ alkanol with.
 84. Theprocess according to claim 77, wherein the alcohol is isopropanol, andthe volume ratio of the nucleic acids-containing sample to isopropanolis 2:1 to 1:1.
 85. The process according to claim 77, wherein themembrane is a hydrophobic membrane.
 86. The process according to claim85, wherein the hydrophobic membrane consists of a polymer with polargroups.
 87. The process according to claim 85, wherein the membrane is ahydrophilic membrane with a hydrophobic surface.
 88. The processaccording to claim 85, characterized in that the membrane consists ofnylon, polyethersulfone, polypropylene, polycarbonate, polyacrylate,acrylic copolymer, polyurethane, polyamide, polyvinylchloride,polyfluorocarbonate, poly-tetrafluoro-ethylene, polyvinylidene fluoride,polyethylene-tetrafluoro-ethylene-copolymerisate, apolyethylene-chlorotrifluoro-ethylene-copolymerisate or, polyphenylenesulfide.
 89. The process according to claim 88, characterized in thatthe membrane consists of a hydrophobic nylon.
 90. The process accordingto claim 87, characterized in that the membrane is coated with awaterproofing agent selected from the group of paraffins, waxes, metalsoaps, optionally containg additives selected from aluminum or zirconiumsalts, quaternary organic compounds, ureic derivates, lipid modifiedmelamine resins, silicones, zinc organic compounds and/or glutaricdialdehyde.
 91. The process according to claim 77, wherein the membraneis a hydrophilic membrane or a hydrophilized membrane.
 92. The processaccording to claim 91, wherein the membrane consists of hydrophilisizednylon, polyethersulfone, polycarbonate, polyacrylate, acrylic copolymer,polyurethane, polyamide, polyvinylchloride, polyfluorocarbonate,poly-tetrafluoro-ethylene, polyvinylidene fluoride,polyethylene-tetrafluoro-ethylene-copolymerisate, apolyethylene-chlorotrifluoroethylene-copolymerisate, cellulose acetate,cellulose nitrate, or polyphenylene sulfide.
 93. The process accordingto claim 92, wherein the membrane consists of cellulose acetate orcellulose nitrate.
 94. The process according to claim 91, wherein themembrane has a pore size of more than 0.45 μm.
 95. The process accordingto claim 91, wherein the membrane has a pore size of more than 0.6 μm.96. An apparatus capable of performing at least one of the steps of theprocess according to any one of claims 1, 9, 14, 21, 29, 30 or 77automatically.
 97. The apparatus according to claim 96, which isequipped with at least one suction mechanism and which performs or isable to perform the addition of buffers and solutions onto the surface.98. An isolation device adapted to the isolation of nucleic acidscomprising: at least one cylindrical upper part with an upper opening, abottom opening, and a membrane located at the bottom opening and fillsthe entire diameter of the upper part; a bottom part containing anabsorbent material; and a mechanism for connecting the upper and bottomparts, such that, after the connection is made, the membrane is incontact with the absorbent material and, in case the connection is notmade, the membrane is not in contact with the absorbent material. 99.The isolation device according to claim 98, characterized in that thebottom part is a cylinder having the same diameter as the upper part.100. The isolation device according to claim 98, characterized in thatthe mechanism for connecting the upper and bottom parts also permits thespatial separation of the upper and bottom parts.
 101. The isolationdevice according to claim 100, wherein the connection mechanism is abayonet socket.
 102. The isolation device according to claim 100,wherein the connection mechanism is a threaded socket.
 103. Theisolation device according to claim 98, characterized in that themechanism for connecting the upper and bottom parts includes a slidingmechanism, which can be slid between the absorbent material and themembrane, to separate the upper and bottom parts.
 104. The isolationdevice according to claim 98, characterized in that the connectionmechanism has a predetermined breaking point between the upper andbottom part.
 105. The isolation device according claim 98, characterizedin that the upper part is a tube, which can be placed in a reactiondevice container.
 106. The isolation device according to claim 98,characterized in that the upper and bottom parts form a tube, which canbe placed in a reaction device container.
 107. The isolation deviceaccording to claim 98, wherein said bottom part is configured to connectwith a plurality of upper parts.
 108. The isolation device according toclaim 98, wherein the absorbent material is a sponge.
 109. The isolationdevice according to claim 98, wherein the absorbent material contains agranulate.
 110. An isolation device adapted for the isolation of nucleicacids comprising: at least one upper part having an upper opening, abottom opening, and a membrane which is located at the bottom openingand which fills the entire diameter of the isolation device; a bottompart having an absorbent material; and a collar surrounding the upperpart at least in the area of the membrane to accommodate a coolant. 111.The isolation device according to claim 110, wherein said collar has twocompartments, which are separated from one another by a frangibleseparation wall; and wherein each of the compartments contains asolution, whereby a coolant is produced when both solutions are mixedafter breaking the separation wall.
 112. A method for isolating nucleicacids comprising contacting a sample containing nucleic acids with ameterial selected from the group of cellulose acetate; non-carboxylized,hydrophobic polyvinylidene fluoride; and massive, hydrophobicpolytetrafluoroethylene.
 113. The method of claim 112, wherein saidmaterial is used in the form of a membrane.
 114. The method of claim112, wherein said material is used in the form of a granulate.
 115. Themethod of claim 112, wherein the material is used in the form of afiber.
 116. The method of claim 115, wherein the fibers are organized asa fleece.
 117. A kit for the isolation of nucleic acids comprising: animmobilization buffer; an elution buffer; and at least one isolationdevice according to one of claims 98 to
 111. 118. The kit of claim 117,characterized in that it also contains a washing buffer.
 119. The kit ofclaim 117 or 118, characterized in that it also contains a lysis buffer.120. The kit according to claim 117 or 118, wherein said at least oneisolation device is configured and instructions are provided forperforming a process according to any one of claims 1, 9, 14, 19, 21,28, 29, 30, or 77.